Treatment of metastatic tumors

ABSTRACT

The present invention is directed to methods and methods for the treatment, inhibition and/or reduction, and detection of metastatic tumors. In some embodiments, the inventive methods include systemic (e.g., intravenous) administration of a chlorotoxin agent that may or may not be labeled. In some embodiments, the inventive methods allow treatment, inhibition and/or reduction, and detection of metastases in the brain. In some embodiments, neovascularization is inhibited and/or newly formed vessels are caused to regress.

RELATED APPLICATIONS

The present application is a Divisional of co-pending U.S. patentapplication Ser. No. 12/466,599, now allowed, which claims priority toand benefit of U.S. provisional application Nos. 61/053,651 (filed onMay 15, 2008), 61/153,273 (filed Feb. 17, 2009), and 61/173,121 (filedApr. 27, 2009) the contents of each of which are herein incorporated byreference in their entirety.

BACKGROUND

The ability of cancer cells to spread, or metastasize, is regarded asthe most deadly aspect of cancer. Cancer cells may break away from aprimary tumor and travel to other parts of the body via the bloodstreamand/or lymphatic system, forming distant metastases. Treatment anddiagnosis of such metastastic tumors presents a challenge, due in partto the number of metastases that can form and the distance from the siteof primary tumor that metastases can travel. The most common sites ofmetastases include the lungs, bone, liver, and brain. Metastases thatlocalize in the brain pose different challenges from those that form inother organs of the body due to the neuroprotective nature of theblood/brain barrier that hinders the delivery of many potentiallyeffective diagnostics and therapeutics to the vasculature and neuraltissue.

Chlorotoxin is a peptide found in venom from the Giant Yellow Israeliscorpion Leiurus Quinqestriatus that has been explored pre-clinically asa candidate for targeting gliomas with 131-iodine (J. A. DeBin et al.,Am. J. Physiol. (Cell Physiol.), 1993, 264, 33: C361-C369; L. Soroceanuet al., Cancer Res., 1998, 58: 4871-4879; S. Shen et al., Neuro-Oncol.,2005, 71: 113-119). Compositions (see U.S. Pat. Nos. 5,905,027 and6,429,187, the contents of each of which are hereby incorporated byreference in their entirety) and methods (see U.S. Pat. Nos. 6,028,174and 6,319,891, the contents of each of which are hereby incorporated byreference in their entirety) for diagnosing and treating neuroectodermaltumors (e.g., gliomas and meningiomas) have been developed based on theability of chlorotoxin to bind to tumor cells of neuroectodermal origin(Soroceanu et al., Cancer Res., 1998, 58: 4871-4879; Ullrich et al.,Neuroreport, 1996, 7: 1020-1024; Ullrich et al., Am. J. Physiol., 1996,270: C1511-C1521).

SUMMARY

The present invention encompasses the finding that chlorotoxin cantarget distant metastases including those found in the brain. Thepresent invention also encompasses the finding that chlorotoxin caninhibit neovascularization and/or cause regression of existing newlyformed blood vessels. Without wishing to be bound by any particulartheory, the inventors propose that the usefulness of chlorotoxin intreating metastatic cancers may be due at least in part to its abilityto inhibit the formation of new blood vessels, and/or to causeregression of newly formed blood vessels, on which metastases arethought to depend.

In one aspect, the invention provides methods for treatment ofmetastatic cancers comprising administering to an individual having orsusceptibe to at least one metastasis, wherein the metastasis arose fromat least one primary tumor, an effective dose of a chlorotoxin agentsuch that the chlorotoxin agent binds to the at least one metastasis. Insome embodiments, the chlorotoxin agent is delivered systemically; insome embodiments, the chlorotoxin agent is delivered intravenously. Insome embodiments, the primary tumor is melanoma, such as cutaneousand/or intraocular melanoma. In some embodiments, the primary tumor isglioma.

In another aspect, the invention provides methods of detecting thepresence of one or more metastases in an individual who has or has hadat least one primary tumor, comprising administering to the individualan effective amount of a labeled chlorotoxin agent and measuring bindingof the labeled chlorotoxin agent in the individual's body. In suchaspects, elevated levels of binding relative to normal (non tumor)tissue in one or more areas of the body other than the site(s) ofprimary tumor(s) is indicative of the presence of one or moremetastases. In some embodiments, administering a second effective amountof a labeled chlorotoxin agent at a second time period and measuringbinding of the labeled chlorotoxin agent in the individual's body allowsassessment of any change in binding (e.g., in extent and/or location ofbinding), which may be indicative of progression, stability, orregression of one or more metastases.

In some embodiments of the methods of treating and/or detectingmetastases, the chlorotoxin agent is delivered systemically. Systemicadministration may comprise intravenous administration. In someembodiments, the chlorotoxin agent binds to at least one tumormetastasis in the brain. In some embodiments, neovascularization isinhibited and/or newly formed blood vessels (which may feed metastases)are caused to regress.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts photomicrographs showing that biotinylated chlorotoxinbinds melanoma cells that have metastasized to the brain.Photomicrographs depict staining of adjacent sections as follows:“TM-601,” section stained with biotinylated chlorotoxin detected by abrown reaction product of DAB with biotin and further counterstainedwith methyl green; “Control,” section stained with only methyl green;and “H&E,” section stained with hematoxylin and eosin.

FIG. 2 depicts photomicrographs showing that biotinylated chlorotoxinbinds melanoma tumor cells that have metastasized to the lung.Photomicrographs depict staining of adjacent sections as follows:“TM-601,” section stained with biotinylated chlorotoxin detected by abrown reaction product of DAB with biotin and further counterstainedwith methyl green; “Control,” section stained with only methyl green;and “H&E,” section stained with hematoxylin and eosin.

FIG. 3 depicts photomicrographs of adjacent sections of normal skinstained as follows: “TM-601,” section stained with biotinylatedchlorotoxin detected by a brown reaction product of DAB with biotin andfurther counterstained with methyl green; “Control,” section stainedwith only methyl green; and “H&E,” section stained with hematoxylin andeosin.

FIG. 4 is the dosing scheme used in the Phase I imaging and safety studyof intravenous ¹³¹I-TM-601 in patients with recurrent or refractorymetastatic solid tumors.

FIG. 5 is a table summarizing the tumor-specific uptake of ¹³¹I-TM-601following intravenous administration in patients with different types ofsolid tumors.

FIG. 6 shows gamma camera images recorded 3 hours, 24 hours, and 7 daysafter intravenous injection of ¹³¹I-TM-601 (30 mCi/0.6 mg) to a patientwith metastatic prostate cancer with known diffuse bone metastases.

FIG. 7 shows gamma camera images recorded 3 hours, 24 hours, and 48hours after intravenous injection of ¹³¹I-TM-601 (30 mCi/0.6 mgto) apatient with metastatic non-small cell lung cancer.

FIG. 8(A) shows a pre-treatment MRI showing a left frontal lesion of apatient with metastatic melanoma (left), and a SPECT image recordedfollowing intravenous injection of ¹³¹I-TM-601 (30 mCi/0.2 mg) to thepatient (right). FIG. 8(B) shows a pre-treatment Magnetic ResonanceImage (MRI) showing the right occipital lesion of a patient withmetastatic melanoma (left), and a SPECT image recorded followingintravenous injection of ¹³¹I-TM-601 (30 mCi/0.2 mg) to the patient(right).

FIG. 9 shows a pre-treatment MRI showing left frontal tumor of a patientwith malignant glioma (left), and SPECT scans taken 48 hours afterintravenous injection of ¹³¹I-TM-601 to the patient (right).

FIG. 10 depicts MRI images from a glioma patient taken before treatment(left panel) and 3 weeks after a dose of 30 mCi of ¹³¹I-TM-601 (rightpanel) delivered systemically. The patient exhibited a significantreduction in enhancing tumor volume and edema.

FIG. 11 depicts MRI images from another glioma patient taken beforetreatment (top panel) and three weeks after a dose of 30 mCi of¹³¹I-TM-601 (bottom panel) delivered systemically. The patient exhibiteda signficant reduction in enhancing tumor volume and edema.

FIG. 12 shows gamma camera images recorded 24 and 48 hours (anterior andposterior views) after intravenous injection of ¹³¹I-TM-601 (30 mCi/0.6mg) to a patient with metastatic melanoma. Uptake of ¹³¹I-TM-601 wasobserved in known distant metastases to the brain, lung, liver, andsubcutaneous nodules.

FIG. 13 depicts results from experiments testing the ability of TM-601to inhibit blood vessel formation in a mouse model of choroidalneovascularization (CNV). Total area of neovascularization (NV) inmm²×10⁻³ is shown for animals receiving either TM-601 or saline vehicle.A statistically significant decrease in choroidal neovascularization wasobserved in animals receiving intraocular injections of 50 μg TM-601 onthe day of disruption of Bruch's membrane and on day 7 (*p<0.05).Choroidal lesions were analyzed on day 14.

FIG. 14 depicts results from experiments testing the ability of TM-601to cause regression of existing neovessels in a mouse model of CNV.Total area of neovascularization (NV) in mm²×10⁻³ is shown for animalsreceiving either TM-601 or saline vehicle. “Baseline” refers to themeasurement taken at day 7 after disruption of Bruch's membrane (i.e.,before treatment with TM-601). A statistically significant regression ofchoroidal neovascularization was observed in animals receivingintraocular injections of 50 μg TM-601 on the day 7 (*p<0.05). For“control” and “TM-601” values, choroidal lesions were analyzed on day14.

FIG. 15 depicts representative microscope images showing thatintravitreal injection with TM-601 led to decreased blood vessels at thesite of laser-induced blood vessels in a mouse model of choroidalneovascularization. Neovascularization was inhibited when TM-601 wasadministered the same day as laser induction (top panel). Existingneovasculature regressed when TM-601 was administered 7 days after laserinduction (bottom panel). On day 14, all mice were perfused withfluorescein-labeled dextran and choroidal flat mounts were prepared andexamined by fluorescence microscopy.

FIG. 16 depicts experimental results showing a Statistically significantdecrease in choroidal neovascularization in animals receiving periocularinjections of 250 μg TM-601 on the day of disruption of Bruch's membraneand on day 7 (*p<0.05). Choroidal lesions were analyzed on day 14.

FIG. 17 depicts experimental results showing dose-dependent inhibitionof choroidal neovascularization with periocular injections of TM-601 ondays 1 and 7 of the study. Choroidal lesions were analyzed on day 14.(*p<0.05).

FIG. 18 depicts experimental results showing statistically significantregression of choroidal neovascularization in animals receivingintravenous injections of TM-601 (3× per week at a dose of 20 mg/kg).Choroidal lesions were analyzed on day 14. (*p<0.05).

FIG. 19 depicts experimental results showing decreased choroidalneovascularization in animals receiving intravenous topical applicationof TM-601 (eye drops 3× per day). Choroidal lesions were analyzed on day14. The difference between areas of NV for eyes that received a dose of0.75 mg/day TM-601 and eyes that received the saline control reached ap-value of 0.059.

FIG. 20 depicts microscope images of frozen sections showinglocalization of TM-601 in CNV areas after intraocular injection ofTM-601. TM-601 was injected on day 7 after laser-induced rupture ofBruch's membrane. Mice were euthanized on day 9. Frozen sections werestained with rabbit anti-TM-601 (red in A, B and C) and with a GSAlectin (green in D, E, and F) to visualize endothelial cells. Eyesinjected with vehicle (B, E, and H) and eyes that were not injected (C,F, and I) did not show positive stained cells within the CNV area. Onthe contrary, sections from eyes treated with TM-601 (A, D, and G)showed prominent staining for TM-601 throughout the area of CNV (A andD). Coregistration of red and green staining is shown on the bottom row(G, H and I). Arrows show the CNV area.

FIG. 21 depicts microscope images of frozen sections showinglocalization of TM-601 in CNV areas after periocular injection ofTM-601. TM-601 was injected, mice were euthanized, and frozen sectionswere stained as described for FIG. 16. (See also Materials and Methodsin Example 6). Staining with rabbit anti-TM-601 is visualized as red inA, B and C and staining with a GSA lectin (to visualize endothelialcells) is visualized as green in D, E, and F. Eyes injected with vehicle(B, E, and H) and eyes that were not injected (C, F, and I) did not showpositive stained cells within the CNV area. On the contrary, sectionsfrom eyes treated with TM-601 (A, D, and G) showed prominent stainingfor TM-601 throughout the area of CNV (A and D). Coregistration of redand green staining is shown on the bottom row (G, H and I). Arrows showthe CNV area.

FIG. 22 depicts microscope images of frozen sections showing the effectof TM-601 on apoptosis within CNV lesions by intraocular injection.TM-601 was injected and mice were euthanized as described for FIG. 16.(See also Materials and Methods in Example 6). Sections were stainedwith nuclear stain (A, B) GSA (C, D) and TUNEL (E, F). Coregistration ofthe three stains is shown on the bottom row (G, H). TUNEL-positive cellswithin the CNV lesions (E) were found in eyes that had intraocularinjections of TM-601 (A, C, E, and G). No TUNEL-positive cells wereobserved in eyes that received injections of vehicle (B, D, F, and H).Arrows show the CNV area.

FIG. 23 depicts microscope images of frozen sections showing the effectof TM-601 on apoptosis within CNV lesions by periocular injection.TM-601 was injected and mice were euthanized as described for FIG. 16.(See also Materials and Methods in Example 6). Sections were stainedwith nuclear stain (A, B), GSA (C, D), and TUNEL (E, F). Coregistrationof the three stains is shown on the bottom row (G, H). TUNEL-positivecells within the CNV lesions (E) were found in eyes that had periocularinjections of TM-601 (A, C, E, and G). No TUNEL-positive cells wereobserved in eyes that received injections of vehicle (B, D, F, and H).Arrows show the CNV area.

FIG. 24 depicts results from experiments measuring HUVEC cellproliferation in the presence of a range of TM-601 concentrations foreither 72 or 120 hrs. Although cell proliferation at higher TM-601concentrations was less than at lower concentration of TM-601, theproliferation rate was not less than that of untreated control cells.

FIG. 25 depicts results from experiments showing co-localization ofTM-601 and Annexin A2 in neovasculature in choroid (induced by laserrupture of Bruch's membrane) and in neovasculature in retina (induced byoxygen-induced ischemia). TM-601 was injected intraocularly andimmunohistochemistry was subsequently performed on tissue sections usinganti-TM-601 antibody and anti-Annexin A2 antibody.

FIG. 26 depicts experimental results showing that TM-601 inhibitsmigration of HUVEC cells and decreases MMP-2 activity. (A) HUVEC cellmigration was stimulated by 50 ng/ml VEGF. Addition of TM-601 inhibitedmigration (as assessed by invasion in a Transwell assay) in adose-dependent manner. Invading cells were visualized on the lowersurface of the Transwell using Giemsa stain. (B) Cell migrationstimulated by either VEGF or bFGF (50 ng/ml) was calculated by visualcell count and TM-601 at 10 μM was shown to inhibit HUVEC invasionthrough the Transwell by approximately 50%. (C) MMP-2 activity in mediataken from cultured HUVEC cells without treatment, treatment with bFGF,or treatment with bFGF together with 10 μM TM-601. Error bars indicatethe standard error.

FIG. 27 depicts experimental results showing that TM-601 decreases MMP-2activity secreted from U87 human glioma cells. MMP-2 activity wasmeasured in media taken from cultured U87 cells without treatment, orwith addition of 10 μM TM-601.

FIG. 28 shows the half-lives of PEGylated chlorotoxin (TM-601-PEG) ascompared to unmodified TM-601 in intravenously injected non-cancerousmice. PEGylation increased the half-life of TM601 by approximately32-fold.

FIG. 29 shows that PEGylated TM-601 can achieve anti-angiogenic effectswith less frequent dosing than unmodified TM-601 in a mouse CNV model.Microvessel density in a CNV model was plotted for various dosingregimens for unmodified TM-601 or for PEGylated TM-601.

DEFINITIONS

Throughout the specification, several terms are employed that aredefined in the following paragraphs.

As used herein, the terms “about” and “approximately,” in reference to anumber, is used herein to include numbers that fall within a range of20%, 10%, 5%, or 1% in either direction (greater than or less than) thenumber unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

As used herein, the term “Annexin A2” refers to a protein product of thegene whose official symbol is ANXA2 (in Homo sapiens) and whose officialfull name is “annexin A2” in the Entrez Gene listing athttp://www.ncbi.nlm.nih.gov. (A variety of sequences for ANXA2transcripts can be found, for example, under GenBank accession nos.M62899, NM_(—)001002857, NM_(—)001002858, NM_(—)004039.) Annexin A2 isalso known, among other things as “annexin II,” and lipocortin 2.

The term “biologically active”, when used herein to characterize apolypeptide, refers to a molecule that shares sufficient amino acidsequence homology with a parent polypeptide to exhibit similar oridentical properties than the polypeptide (e.g., ability to specificallybind to cancer cells and/or to be internalized into cancer cells and/orto kill cancer cells).

As used herein, the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancers include, but are notlimited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticularly, examples of such cancers include lung cancer, bone cancer,liver cancer, pancreatic cancer, skin cancer, cancer of the head orneck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the sexual and reproductiveorgans, Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, and pituitary adenoma.

As used herein, the term “cancer cell” refers to a cell in a mammal(e.g., a human being) in vivo which undergoes undesired and unregulatedcell growth or abnormal persistence or abnormal invasion of tissues. Invitro, this term also refers to a cell line that is a permanentlyimmortalized established cell culture that will proliferate indefinitelyand in an unregulated manner given appropriate fresh medium and space.

As used herein, the term “cancer patient” can refer to an individualsuffering from or susceptible to cancer. A cancer patient may or may nothave been diagnosed with cancer. The term also includes individuals thathave previously undergone therapy for cancer.

The terms “chemotherapeutics” and “anti-cancer agents or drugs” are usedherein interchangeably. They refer to those medications that are used totreat cancer or cancerous conditions. Anti-cancer drugs areconventionally classified in one of the following group: alkylatingagents, purine antagonists, pyrimidine antagonists, plant alkaloids,intercalating antibiotics, aromatase inhibitors, anti-metabolites,mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors,enzymes, topoisomerase inhibitors, biological response modifiers,anti-hormones and anti-androgens. Examples of such anti-cancer agentsinclude, but are not limited to, BCNU, cisplatin, gemcitabine,hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil,vincristine, vinblastine, procarbazine, decarbazine, altretamine,methotrexate, mercaptopurine, thioguanine, fludarabine phosphate,cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide,irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin,idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide,leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine,asparaginase, mitoxantrone, mitotane and amifostine.

The term “combination therapy”, as used herein, refers to thosesituations in which two or more different pharmaceutical agents areadministered in overlapping regimens so that the subject issimultaneously exposed to both agents.

As used herein, the abbreviation “CTCAE” refers to Common TerminologyCriteria for Adverse Events, a scale developed by the National CancerInstitutes for adverse event (AE) description and grading that iscommonly used in clinical trials.

The term “cytotoxic”, when used herein to characterize a moiety,compound, drug or agent refers to a moiety, compound, drug or agent thatinhibits or prevents the function of cells and/or causes destruction ofcells.

A “dosing regimen”, as that term is used herein, refers to a set of unitdoses (typically more than one) that are administered individuallyseparated by periods of time. The recommended set of doses (i.e.,amounts, timing, route of administration, etc.) for a particularpharmaceutical agent constitutes its dosing regimen.

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Forexample, in certain embodiments of the present invention, the purpose(s)may be: to specifically bind to a target tissue, to slow down or stopthe progression, aggravation, or deterioration of the symptoms of acancer, to bring about amelioration of the symptoms of the cancer,and/or to cure the cancer. The relevant intended purpose may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). A therapeuticallyeffective amount is commonly administered in a dosing regimen that maycomprise multiple unit doses. For any particular pharmaceutical agent, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents. Insome embodiments, the specific therapeutically effective amount (and/orunit dose) for any particular patient may depend upon a variety offactors including the disorder being treated and the severity of thedisorder; the activity of the specific pharmaceutical agent employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and/or rate of excretion or metabolism of the specificpharmaceutical agent employed; the duration of the treatment; and likefactors as is well known in the medical arts.

As used herein, terms “fluorophore”, “fluorescent moiety”, “fluorescentlabel”, “fluorescent dye” and “fluorescent labeling moiety” are usedherein interchangeably. They refer to a molecule that, in solution andupon excitation with light of appropriate wavelength, emits light back.Numerous fluorescent dyes of a wide variety of structures andcharacteristics are suitable for use in the practice of this invention.Similarly, methods and materials are known for fluorescently labelingnucleic acids (see, for example, R. P. Haugland, “Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals 1992-1994”, 5^(th)Ed., 1994, Molecular Probes, Inc.). In choosing a fluorophore, it isoften desirable that the fluorescent molecule absorbs light and emitsfluorescence with high efficiency (i.e., high molar absorptioncoefficient and fluorescence quantum yield, respectively) and isphotostable (i.e., it does not undergo significant degradation uponlight excitation within the time necessary to perform the analysis).

As used herein, the term “fusion protein” refers to a moleculecomprising two or more proteins or fragments thereof linked by acovalent bond via their individual peptide backbones, most preferablygenerated through genetic expression of a polynucleotide moleculeencoding those proteins.

As used herein, the term “homologous” (or “homology”) refers to a degreeof identity between two polypeptide molecules or between two nucleicacid molecules. When a position in both compared sequences is occupiedby the same base or amino acid monomer subunit, then the respectivemolecules are homologous at that position. The percentage of homologybetween two sequences corresponds to the number of matching orhomologous positions shared by the two sequences divided by the numberof positions compared and multiplied by 100. Generally, a comparison ismade when two sequences are aligned to give maximum homology. Homologousamino acid sequences share identical or similar amino acid residues.Similar residues are conservative substitutions for, or “allowed pointmutations” of, corresponding amino acid residues in a referencesequence. “Conservative substitutions” of a residue in a referencesequence are substitutions that are physically or functionally similarto the corresponding reference residue, e.g., that have a similar size,shape, electric charge, chemical properties, including the ability toform covalent or hydrogen bonds, or the like. In some embodiments,conservative substitutions utilized in accordance with the presentinvention are those fulfilling the criteria defined for an “acceptedpoint mutation” by Dayhoff et al. (“Atlas of Protein Sequence andStructure”, 1978, Nat. Biomed. Res. Foundation, Washington, D.C., Suppl.3, 22: 354-352).

The terms “individual” and “subject” are used herein interchangeably.They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog,cat, cattle, swine, sheep, horse or primate) that can be afflicted withor is susceptible to a disease or disorder (e.g., cancer) but may or maynot have the disease or disorder. In many embodiments, the subject is ahuman being. In many embodiments, the subject is a patient. Unlessotherwise stated, the terms “individual” and “subject” do not denote aparticular age, and thus encompass adults, children, and newborns.

As used herein, the term “inhibit” means to prevent something fromhappening, to delay occurrence of something happening, and/or to reducethe extent or likelihood of something happening. Thus, “inhibitingmetastases” and “inhibiting the formation of metastases” is intended toencompass preventing, delaying, and/or reducing the likelihood ofoccurrence of metastases as well as reducing the number, growth rate,size, etc., of metastases.

As used herein, the term “initiation” when applied to a dosing regimencan be used to refer to a first administration of a pharmaceutical agentto a subject who has not previously received the pharmaceutical agent.Alternatively or additionally, the term “initiation” can be used torefer to administration of a particular unit dose of a pharmaceuticalagent during therapy of a patient.

The terms “labeled” and “labeled with a detectable agent or moiety” areused herein interchangeably to specify that an entity (e.g., achlorotoxin or chlorotoxin conjugate) can be visualized, for examplefollowing binding to another entity (e.g., a neoplastic tumor tissue).Preferably the detectable agent or moiety is selected such that itgenerates a signal which can be measured and whose intensity is relatedto (e.g., proportional to) the amount of bound entity. A wide variety ofsystems for labeling and/or detecting proteins and peptides are known inthe art. Labeled proteins and peptides can be prepared by incorporationof, or conjugation to, a label that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical,chemical or other means. A label or labeling moiety may be directlydetectable (i.e., it does not require any further reaction ormanipulation to be detectable, e.g., a fluorophore is directlydetectable) or it may be indirectly detectable (i.e., it is madedetectable through reaction or binding with another entity that isdetectable, e.g., a hapten is detectable by immunostaining afterreaction with an appropriate antibody comprising a reporter such as afluorophore). Suitable detectable agents include, but are not limitedto, radionuclides, fluorophores, chemiluminescent agents,microparticles, enzymes, colorimetric labels, magnetic labels, haptens,Molecular Beacons, aptamer beacons, and the like.

As used herein, the term “metastasis” (sometimes abbreviated as “mets;”plural “metastases”) refers to the spread of tumor cells from one organor tissue to another location. The term also refers to tumor tissue thatforms in a new location as a result of metastasis. A “metastatic cancer”is a cancer that spreads from its original, or primary, location, andmay also be referred to as a “secondary cancer” or “secondary tumor.”Generally, metastastic tumors are named for the tissue of the primarytumor from which they originate. Thus, a breast cancer that hasmetastasized to the lung may be referred to as “metastatic breastcancer” even though some cancer cells are located in the lung.

As used herein, the term “neovasculature” refers to newly formed bloodvessels that have not yet fully matured, i.e., do not have a fullyformed endothelial lining with tight cellular junctions or a completelayer of surrounding smooth muscle cells. As used herein, the term“neovessel” is used to refer to a blood vessel in neovasculature.

The terms “normal” and “healthy” are used herein interchangeably. Theyrefer to an individual or group of individuals who do not have a tumor.The term “normal” is also used herein to qualify a tissue sampleisolated from a healthy individual.

The terms “pharmaceutical agent”, “therapeutic agent” and “drug” areused herein interchangeably. They refer to a substance, molecule,compound, agent, factor or composition effective in the treatment,inhibition, and/or detection of a disease, disorder, or clinicalcondition.

A “pharmaceutical composition” is herein defined as a composition thatcomprises an effective amount of at least one active ingredient (e.g., achlorotoxin or chlorotoxin conjugate that may or may not be labeled),and at least one pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium which does not interfere with the effectiveness of thebiological activity of the active ingredient(s) and which is notexcessively toxic to the host at the concentration at which it isadministered. The term includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art (see forexample, “Remington's Pharmaceutical Sciences”, E. W. Martin, 18^(th)Ed., 1990, Mack Publishing Co.: Easton, Pa., which is incorporatedherein by reference in its entirety).

As used herein, the term “primary tumor” refers to a tumor that is atthe original site where the tumor first arose, i.e., as opposed tohaving spread there.

The terms “protein”, “polypeptide”, and “peptide” are used hereininterchangeably, and refer to amino acid sequences of a variety oflengths, either in their neutral (uncharged) forms or as salts, andeither unmodified or modified by glycosylation, side chain oxidation, orphosphorylation. In certain embodiments, the amino acid sequence is thefull-length native protein. In other embodiments, the amino acidsequence is a smaller fragment of the full-length protein. In stillother embodiments, the amino acid sequence is modified by additionalsubstituents attached to the amino acid side chains, such as glycosylunits, lipids, or inorganic ions such as phosphates, as well asmodifications relating to chemical conversion of the chains, such asoxidation of sulfhydryl groups. Thus, the term “protein” (or itsequivalent terms) is intended to include the amino acid sequence of thefull-length native protein, subject to those modifications that do notchange its specific properties. In particular, the term “protein”encompasses protein isoforms, i.e., variants that are encoded by thesame gene, but that differ in their pI or MW, or both. Such isoforms candiffer in their amino acid sequence (e.g., as a result of alternativeslicing or limited proteolysis), or in the alternative, may arise fromdifferential post-translational modification (e.g., glycosylation,acylation or phosphorylation).

The term “protein analog”, as used herein, refers to a polypeptide thatpossesses a similar or identical function as a parent polypeptide butneed not necessarily comprise an amino acid sequence that is similar oridentical to the amino acid sequence of the parent polypeptide, orpossess a structure that is similar or identical to that of the parentpolypeptide. Preferably, in the context of the present invention, aprotein analog has an amino acid sequence that is at least 30% (morepreferably, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 99%)identical to the amino acid sequence of the parent polypeptide.Moreover, those of ordinary skill in the art will understand thatprotein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90%, 96%, 97%,98% or 99% in one or more highly conserved regions usually encompassingat least 3-4 and often up to 20 or more amino acids, with the parentpolypeptide, is encompassed in the term “protein analog).

As used herein, the term “protein fragment” refers to a polypeptidecomprising an amino acid sequence of at least 5 amino acid residues ofthe amino acid sequence of a second polypeptide. A fragment of a proteinmay or may not possess a functional activity of the parent polypeptide.

The term “regress,” when used to refer to blood vessels and/orvasculature (including neovasculature and/or neovessels), is used hereinto mean to retract, shrink, etc.

As used herein, the term “small molecule” includes any chemical or othermoiety that can act to affect biological processes. Small molecules caninclude any number of therapeutic agents presently known and used, orcan be small molecules synthesized in a library of such molecules forthe purpose of screening for biological function(s). Small molecules aredistinguished from macromolecules by size. Small molecules suitable foruse in the present invention usually have molecular weight less thanabout 5,000 daltons (Da), preferably less than about 2,500 Da, morepreferably less than 1,000 Da, most preferably less than about 500 Da.

As used herein, the term “susceptible” means having an increased riskfor and/or a propensity for (typically based on genetic predisposition,environmental factors, personal history, or combinations thereof)something, i.e., a disease, disorder, or condition such as metastaticcancer, than is observed in the general population. The term takes intoaccount that an individual “susceptible” for a condition may never bediagnosed with the condition.

As used herein, the term “systemic administration” refers toadministration of an agent such that the agent becomes widelydistributed in the body in significant amounts and has a biologicaleffect, e.g., its desired effect, in the blood and/or reaches itsdesired site of action via the vascular system. Typical systemic routesof administration include administration by (1) introducing the agentdirectly into the vascular system or (2) oral, pulmonary, orintramuscular administration wherein the agent is adsorbed, enters thevascular system, and is carried to one or more desired site(s) of actionvia the blood.

The term “tissue” is used herein in its broadest sense. A tissue may beany biological entity that can (but does not necessarily) comprise atumor cell. In the context of the present invention, in vitro, in vivoand ex vivo tissues are considered. Thus, a tissue may be part of anindividual or may be obtained from an individual (e.g., by biopsy).Tissues may also include sections of tissue such as frozen sectionstaken for histological purposes or archival samples with knowndiagnosis, treatment and/or outcome history. The term tissue alsoencompasses any material derived by processing the tissue sample.Derived materials include, but are not limited to, cells (or theirprogeny) isolated from the tissue. Processing of the tissue sample mayinvolve one or more of: filtration, distillation, extraction,concentration, inactivation of interfering components, addition ofreagents, and the like.

The term “treatment” is used herein to characterize a method or processthat is aimed at (1) delaying or preventing the onset of a disease,disorder, or condition; (2) slowing down or stopping the progression,aggravation, or deterioration of one or more symptoms of the disease,disorder, or condition; (3) bringing about ameliorations of the symptomsof the disease, disorder, or condition; (4) reducing the severity orincidence of the disease, disorder, or condition; or (5) curing thedisease, disorder., or condition. A treatment may be administered priorto the onset of the disease, disorder, or condition, for a prophylacticor preventive action. Alternatively or additionally, the treatment maybe administered after initiation of the disease, disorder, or condition,for a therapeutic action.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

As already mentioned above, the present invention is directed to methodsfor the treatment and/or detection of tumor metastases. Methods providedherein generally comprise administration of a chlorotoxin agent that mayor may not be labeled with a detectable moiety. In certain embodiments,the chlorotoxin agent binds to tumor metastases. In certain embodiments,the chlorotoxin agent inhibits and/or reduces the likelihood offormation of new metastases. In certain embodiments, the chlorotoxinagent is administered systemically (e.g., intravenously) and/or thechlorotoxin agent crosses the blood/brain barrier. Thus, in someembodiments, the invention provides methods of treating, inhibiting,and/or detecting metastases located in the brain. In certainembodiments, the formation of new blood vessels is inhibited and/orexisting neovasculature regresses.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual”, 1982; “DNA Cloning: APractical Approach,” Volumes I and II, D. N. Glover (Ed.), 1985;“Oligonucleotide Synthesis”, M. J. Gait (Ed.), 1984; “Nucleic AcidHybridization”, B. D. Hames & S. J. Higgins (Eds.), 1985; “Transcriptionand Translation” B. D. Hames & S. J. Higgins (Eds.), 1984; “Animal CellCulture”, R. I. Freshney (Ed.), 1986; “Immobilized Cells And Enzymes”,IRL Press, 1986; B. Perbal, “A Practical Guide To Molecular Cloning”,1984.

I. Chlorotoxin Agents

Methods of treatment and/or detection of the present invention involveadministering, to an individual in need thereof (such as, for anexample, an individual who has, has had, is at risk of developing,and/or susceptible to at least one metastasis), an effective dose of atleast one chlorotoxin agent such that the chlorotoxin agent binds to theat least one metastasis. As used herein, the term “chlorotoxin agent”refers to a compound that comprises at least one chlorotoxin moiety. Incertain embodiments, a chlorotoxin agent comprises at least onechlorotoxin moiety associated with at least one therapeutic moiety(e.g., an anti-cancer agent). The chlorotoxin moiety (and/or therapeuticmoiety) may be associated with at least one labeling moiety.

A. Chlorotoxin Moieties

As used herein, the term “chlorotoxin moiety” refers to a chlorotoxin, abiologically active chlorotoxin subunit or a chlorotoxin derivative.

In certain embodiments, the term “chlorotoxin” refers to thefull-length, 36 amino acid polypeptide naturally derived from Leiurusquinquestriatus scorpion venom (DeBin et al., Am. J. Physiol., 1993,264: C361-369), which comprises the amino acid sequence of nativechlorotoxin as set forth in SEQ ID NO. 1 of International ApplicationNo. WO 2003/101474, the contents of which are incorporated herein byreference. The term “chlorotoxin” includes polypeptides comprising SEQID NO. 1 which have been synthetically or recombinantly produced, suchas those disclosed in U.S. Pat. No. 6,319,891 (which is incorporatedherein by reference in its entirety). The sequences referred to hereinare provided as a computer readable form of Sequence Listing(ST.25.txt).

A “biologically active chlorotoxin subunit” is a peptide comprising lessthan the 36 amino acids of chlorotoxin and which retains at least oneproperty or function of chlorotoxin. As used herein, a “property orfunction” of chlorotoxin includes, but is not limited to, the ability toarrest abnormal cell growth; ability to specifically bind to atumor/cancer cell compared to a normal cell; ability to specificallybind to a metastasizing tumor/cancer cell or a tumor/cancer cell in ametastasis compared to a normal cell; ability to be internalized into atumor/cancer cell; ability to kill a tumor/cancer cell; and/or abilityto suppress formation of and/or cause regression of neovessels. Thetumor/cancer cell may be in vitro, ex vivo, in vitro, part of ametastasis, a primary isolate from a subject, a cultured cell, or a cellline.

As used herein, the term “biologically active chlorotoxin derivative”refers to any of a wide variety of derivatives, analogs, variants,polypeptide fragments and mimetics of chlorotoxin and related peptideswhich retain at least one property or function of chlorotoxin (asdescribed above). Examples of chlorotoxin derivatives include, but arenot limited to, peptide variants of chlorotoxin, peptide fragments ofchlorotoxin, for example, fragments comprising or consisting ofcontiguous 10-mer peptides of SEQ ID No. 1, 2, 3, 4, 5, 6, or 7 as setforth in International Application No. WO 2003/101474 or comprisingresidues 10-18 or 21-30 of SEQ ID No. 1 as set forth in InternationalApplication No. WO 2003/101474, core binding sequences, and peptidemimetics.

Examples of chlorotoxin derivatives include peptides having a fragmentof the amino acid sequence set forth in SEQ ID No. 1 of InternationalApplication No. WO 2003/101474, having at least about 7, about 8, about9, about 10, about 15, about 20, about 25, about 30 or about 35contiguous amino acid residues, associated with the activity ofchlorotoxin. Such fragments may contain functional regions of thechlorotoxin peptide, identified as regions of the amino acid sequencethat correspond to known peptide domains, as well as regions ofpronounced hydrophilicity. Such fragments may also include two coresequences linked to one another, in any order, with intervening aminoacid removed or replaced by a linker.

Derivatives of chlorotoxin include polypeptides comprising aconservative or non-conservative substitution of at least one amino acidresidue when the derivative sequence and the chlorotoxin sequence aremaximally aligned. The substitution may be one that enhances at leastone property or function of chlorotoxin, inhibits at least one propertyor function of chlorotoxin, or is neutral to at least one property orfunction of chlorotoxin.

Examples of derivatives of chlorotoxin suitable for use in the practiceof the present invention are described in International Application No.WO 2003/101474 (which is incorporated herein by reference in itsentirety). Particular examples include polypeptides that comprise orconsist of SEQ ID NO. 8 or SEQ ID NO. 13 as set forth in thisInternational Application, as well as variants, analogs, and derivativesthereof.

Other examples of chlorotoxin derivatives include those polypeptidescontaining pre-determined mutations by, e.g., homologous recombination,site-directed or PCR mutagenesis, and the alleles or othernaturally-occurring variants of the family of peptides; and derivativeswherein the peptide has been covalently modified by substitution,chemical, enzymatic or other appropriate means with a moiety other thana naturally-occurring amino acid (for example a detectable moiety suchas enzyme or a radioisotope).

Chlorotoxin and peptide derivatives thereof can be prepared using any ofa wide variety of methods, including standard solid phase (or solutionphase) peptide synthesis methods, as is known in the art. In addition,the nucleic acids encoding these peptides may be synthesized usingcommercially available oligonucleotide synthesis instrumentation and theproteins may be produced recombinantly using standard recombinantproduction systems.

Other suitable chlorotoxin derivatives include peptide mimetics thatmimic the three-dimensional structure of chlorotoxin. Such peptidemimetics may have significant advantages over naturally occurringpeptides including, for example, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc), altered specificity (e.g.,broad-spectrum biological activities, reduced antigenicity and others).

In certain embodiments, mimetics are molecules that mimic elements ofchlorotoxin peptide secondary structure. Peptide backbone of proteinsexists mainly to orient amino acid side chains in such a way as tofacilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is expected to permit molecular interactionssimilar to the natural molecule. Peptide analogs are commonly used inthe pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of compounds arealso referred to as peptide mimetics or peptidomimetics (see, forexample, Fauchere, Adv. Drug Res., 1986, 15: 29-69; Veber & Freidinger,1985, Trends Neurosci., 1985, 8: 392-396; Evans et al., J. Med. Chem.,1987, 30: 1229-1239) and are usually developed with the aid ofcomputerized molecular modeling.

Generally, peptide mimetics are structurally similar to a paradigmpolypeptide (i.e., a polypeptide that has a biochemical property orpharmacological activity), but have one or more peptide linkagesoptionally replaced by a non-peptide linkage. The use of peptidemimetics can be enhanced through the use of combinatorial chemistry tocreate drug libraries. The design of peptide mimetics can be aided byidentifying amino acid mutations that increase or decrease the bindingof a peptide to, for example, a tumor cell. Approaches that can be usedinclude the yeast two hybrid method (see, for example, Chien et al.,Proc. Natl. Acad. Sci. USA, 1991, 88: 9578-9582) and using the phasedisplay method. The two-hybrid method detects protein-proteininteractions in yeast (Field et al., Nature, 1989, 340: 245-246). Thephage display method detects the interaction between an immobilizedprotein and a protein that is expressed on the surface of phages such aslambda and M13 (Amberg et al., Strategies, 1993, 6: 2-4; Hogrefe et al.,Gene, 1993, 128: 119-126). These methods allow positive and negativeselection of peptide-protein interactions and the identification of thesequences that determine these interactions.

In certain embodiments, a chlorotoxin agent comprises a polypeptidetoxin of another scorpion species that displays similar or relatedactivity to chlorotoxin described above. As used herein, the term“similar or related activity to chlorotoxin” refers, in particular, tothe selective/specific binding to tumor/cancer cells. Examples ofsuitable related scorpion toxins include, but are not limited to toxinsor related peptides of scorpion origin that display amino acid and/ornucleotide sequence identity to chlorotoxin. Examples of relatedscorpion toxins include, but are not limited to, CT neurotoxin fromMesobuthus martenssi (GenBank Accession No. AAD473730), Neurotoxin BmK41-2 from Buthus martensii karsch (GenBank Accession No. A59356),Neurotoxin Bm12-b from Buthus martensii (GenBank Accession No.AAK16444), Probable Toxin LGH 8/6 from Leiurus quinquestriatus hebraeu(GenBank Accession No. P55966), and Small toxin from Mesubutus tamulussindicus (GenBank Accession No. P15229).

Related scorpion toxins suitable for use in the present inventioncomprise polypeptides that have an amino acid sequence of at least about75%, at least about 85%, at least about 90%, at least about 95%, or atleast about 99% sequence identity with the entire chlorotoxin sequenceas set forth in SEQ ID No. 1 of International Application No. WO2003/101474 (which is incorporated herein by reference in its entirety).In certain embodiments, related scorpion toxins include those scorpiontoxins that have a sequence homologous to SEQ ID NO. 8 or SEQ ID NO. 13of chlorotoxin, as set forth in International Application No. WO2003/101474.

In certain embodiments, a chlorotoxin moiety within a chlorotoxin agentis labeled. Examples of labeling methods and labeling moieties aredescribed below.

B. Therapeutic Moieties

As already mentioned above, in certain embodiments, a chlorotoxin agentcomprises at least one chlorotoxin moiety associated with at least onetherapeutic moiety. Suitable therapeutic moieties include any of a largevariety of substances, molecules, compounds, agents or factors that areeffective in the treatment of a disease or clinical condition. Incertain embodiments, a therapeutic moiety is a chemotherapeutic (i.e.,an anti-cancer drug). Suitable anti-cancer drugs include any of a largevariety of substances, molecules, compounds, agents or factors that aredirectly or indirectly toxic or detrimental to cancer cells.

As will be appreciated by one of ordinary skill in the art, atherapeutic moiety may be a synthetic or natural compound: a singlemolecule, a mixture of different molecules or a complex of differentmolecules. Suitable therapeutic moieties can belong to any of a varietyof classes of compounds including, but not limited to, small molecules,peptides, proteins, saccharides, steroids, antibodies (includingfragments and variants thereof), fusion proteins, antisensepolynucleotides, ribozymes, small interfering RNAs, peptidomimetics,radionuclides, and the like.

When a therapeutic moiety comprises an anti-cancer drug, the anti-cancerdrug can be found, for example, among the following classes ofanti-cancer drugs: alkylating agents, anti-metabolic drugs, anti-mitoticantibiotics, alkaloidal anti-tumor agents, hormones and anti-hormones,interferons, non-steroidal anti-inflammatory drugs, and various otheranti-tumor agents such as kinase inhibitors, proteasome inhibitors andNF-κB inhibitors.

Examples of anti-cancer drugs include, but are not limited to,alkylating drugs (e.g., mechlorethamine, chlorambucil, cyclophosphamide,melphalan, ifosfamide, temozolomide, etc.), antimetabolites (e.g.,methotrexate, etc.), purine antagonists and pyrimidine antagonists(e.g., 6-mercaptopurine, 5-fluorouracil, cytraribine, gemcitabine,etc.), spindle poisons (e.g., vinblastine, vincristine, vinorelbine,paclitaxel, etc.), podophyllotoxins (e.g., etoposide, irinotecan,topotecan, etc.), antibiotics (e.g., doxorubicin, bleomycin, mitomycin,etc.), nitrosureas (e.g., carmustine, lomustine, nomustine, etc.),inorganic ions (e.g., cisplatin, carboplatin, etc.), enzymes (e.g.,asparaginase, etc.), and hormones (e.g., tamoxifen, leuprolide,flutamide, megestrol, etc.), to name a few. For a more comprehensivediscussion of updated cancer therapies see, http://www.cancer.gov/, alist of the FDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Some anti-cancer drugs act by arresting the growth and/or replication ofcancer cells. Such drugs are generally classified as “cytostatic.” Incertain embodiments, a therapeutic moiety comprises a cytostatic agent.Examples of cytostatic agents includealkylating agents,anti-metabolites, plant alkyloids and terpenoids (including vincaalkaloids, podophyllotoxin, taxanes, etc.; VP-16 is an example of aplant alkaloid), topoisomerase inhibitors, antitumor antibodies,hormones, etc.

In certain embodiments, a therapeutic moiety comprises a cytotoxicagent. Examples of cytotoxic agents include toxins, other bioactiveproteins, conventional chemotherapeutic agents, enzymes, andradioisotopes.

Examples of suitable cytotoxic toxins include, but are not limited to,bacterial and plant toxins such as gelonin, ricin, saponin, Pseudomonasexotoxin, pokeweed antiviral protein, diphtheria toxin, etc.

Examples of suitable cytotoxic bioactive proteins include, but are notlimited to, proteins of the complement system (or complement proteins).The complement system is a complex biochemical cascade that helps clearpathogens from an organism, and promotes healing (B. P. Morgan, Crit.Rev. Clin. Lab. Sci., 1995, 32: 265). The complement system consists ofmore than 35 soluble and cell-bound proteins, 12 of which are directlyinvolved in the complement pathways.

Examples of suitable cytotoxic chemotherapeutic agents include, but arenot limited to, taxanes (e.g., docetaxel, paclitaxel, etc.),maytansines, duocarmycins, CC-1065, auristatins, calicheamincins andother enediyne anti-tumor antibiotics. Other examples include theanti-folates (e.g., aminopterin, methotrexate, pemetrexed, raltitrexed,etc.), vinca alkaloids (e.g., vincristine, vinblastine, etoposide,vindesine, vinorelbine, etc.), and anthracyclines (e.g., daunorubicin,doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, etc.).

Examples of suitable cytotoxic enzymes include, but are not limited to,nucleolytic enzymes.

Examples of suitable cytotoxic radioisotopes include any α-, β- orγ-emitter which, when localized at a tumor site, results in celldestruction (S. E. Order, “Analysis, Results, and Future Prospective ofthe Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”,Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin etal. (Eds.), Academic Press, 1985). Examples of such radioisotopesinclude, but are not limited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), astatine-211 (²¹¹At),rhenium-186 (¹⁸⁶Re), rhenium-186 (¹⁸⁸Re), phosphorus-32 (³²P),yttrium-90 (⁹⁰Y), samarium-153 (¹⁵³Sm), and lutetium-177 (¹¹⁷Lu).

Alternatively or additionally, therapeutic moieties suitable for use inthe present invention may be any of the therapeutic moieties describedin co-owned provisional applications entitled “Chlorotoxins as DrugCarriers” (U.S. Ser. No. 60/954,409) filed on Aug. 7, 2007 and “SystemicAdministration of Chlorotoxin Agents for the Diagnosis and Treatment ofTumors” (USSN 60/) filed on Oct. 12, 2007, the entire contents of whichare incorporated herein by reference in their entirety. Examples ofclasses of such therapeutic moieties include, but are not limited to,poorly water soluble anti-cancer agents, anti-cancer agents associatedwith drug resistance, antisense nucleic acids, ribozymes, triplexagents, short-interfering RNAs (siRNAs), photosensitizers,radiosensitizers, superantigens, prodrug activating enzymes, andanti-angiogenic agents.

In certain embodiments, a therapeutic (e.g., anti-cancer) agent within achlorotoxin agent is a nucleic acid agent.

Numerous cancers and tumors have been shown to be associated withvarying degrees of genetic impairment, such as point mutations, genedeletions, or duplications. Many new strategies for the treatment ofcancer, such as “antisense”, “antigene”, and “RNA interference” havebeen developed to modulate the expression of genes (A. Kalota et al.,Cancer Biol. Ther., 2004, 3: 4-12; Y. Nakata et al., Crit. Rev.Eukaryot. Gene Expr., 2005, 15: 163-182; V. Wacheck and U.Zangmeister-Wittke, Crit. Rev. Oncol. Hematol., 2006, 59: 65-73; A.Kolata et al., Handb. Exp. Pharmacol., 2006, 173: 173-196). Theseapproaches utilize, for example, antisense nucleic acids, ribozymes,triplex agents, or short interfering RNAs (siRNAs) to block thetranscription or translation of a specific mRNA or DNA of a target gene,either by masking that mRNA with an antisense nucleic acid or DNA with atriplex agent, by cleaving the nucleotide sequence with a ribozyme, orby destruction of the mRNA, through a complex mechanism involved inRNA-interference. In all of these strategies, mainly oligonucleotidesare used as active agents, although small molecules and other structureshave also been applied. While the oligonucleotide-based strategies formodulating gene expression have a huge potential for the treatment ofsome cancers, pharmacological applications of oligonucleotides have beenhindered mainly by the ineffective delivery of these compounds to theirsites of action within cancer cells. (P. Herdewijn et al., AntisenseNucleic Acids Drug Dev., 2000, 10: 297-310; Y. Shoji and H. Nakashima,Curr. Charm. Des., 2004, 10: 785-796; A. W Tong et al., Curr. Opin. Mol.Ther., 2005, 7: 114-124).

Chlorotoxin agents are provided herein that comprise a toxin moiety(e.g., chlorotoxin moiety) and a nucleic acid molecule that is useful asa therapeutic (e.g., anti-cancer) agent. A variety of chemical types andstructural forms of nucleic acid can be suitable for such strategies.These include, by way of non-limiting example, DNA, includingsingle-stranded (ssDNA) and double-stranded (dsDNA); RNA, including, butnot limited to ssRNA, dsRNA, tRNA, mRNA, rRNA, enzymatic RNA; RNA:DNAhybrids, triplexed DNA (e.g., dsDNA in association with a shortoligonucleotide), and the like.

In some embodiments of the present invention, the nucleic acid agentpresent in a chlorotoxin agent is between about 5 and 2000 nucleotideslong. In some embodiments, the nucleic acid agent is at least about 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more nucleotides long. In someembodiments, the nucleic acid agent is less than about 2000, 1900, 1800,1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500,450, 400, 350, 300, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20 orfewer nucleotides long.

In some embodiments, a nucleic acid agent present in a chlorotoxin agentof the present invention comprises a promoter and/or other sequencesthat regulate transcription. In some embodiments, a nucleic acid agentpresent in a chlorotoxin agent of the present invention comprises anorigin of replication and/or other sequences that regulate replication.In some embodiments, a nucleic acid agent present in a chlorotoxin agentof the present invention does not include a promoter and/or an origin ofreplication.

Nucleic acid anti-cancer agents suitable for use in the practice of thepresent invention include those agents that target genes associated withtumorigenesis and cell growth or cell transformation (e.g.,proto-oncogenes, which code for proteins that stimulate cell division),angiogenic/anti-angiogenic genes, tumor suppressor genes (which code forproteins that suppress cell division), genes encoding proteinsassociated with tumor growth and/or tumor migration, and suicide geneswhich induce apoptosis or other forms of cell death, especially suicidegenes that are most active in rapidly dividing cells.

Examples of gene sequences associated with tumorigenesis and/or celltransformation include MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1,TLS-FUS, PAX3-FKHR, Bc1-2, AML1-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC,NF-1, Rb, p53, MDM2 and the like; overexpressed sequences such asmultidrug resistance genes; cyclins; beta-Catenin; telomerase genes;c-myc, n-myc, Bc1-2, Erb-B1 and Erb-B2; and mutated sequences such asRas, Mos, Raf, and Met. Examples of tumor suppressor genes include, butare not limited to, p53, p21, RB1, WT1, NF1, VHL, APC, DAP kinase, p16,ARF, Neurofibromin, and PTEN. Examples of genes that can be targeted bynucleic acid molecules useful in anti-cancer therapy include genesencoding proteins associated with tumor migration such as integrins,selectins and metalloproteinases; anti-angiogenic genes encodingproteins that promote the formation of new vessels such as VascularEndothelial Growth Factor (VEGF) or VEGFr; anti-angiogenic genesencoding proteins that inhibit neovascularization such as endostatin,angiostatin, and VEGF-R2; and genes encoding proteins such asinterleukins, interferon, fibroblast growth factor (α-FGF and β-FGF),insulin-like growth factor (e.g., IGF-1 and IGF-2), Platelet-derivedgrowth factor (PDGF), tumor necrosis factor (TNF), Transforming GrowthFactor (e.g., TGF-α and TGF-β), Epidermal growth factor (EGF),Keratinocyte Growth Factor (KGF), stem cell factor and its receptorc-Kit (SCF/c-Kit) ligand, CD40L/CD40, VLA-4 VCAM-1, ICAM-1/LFA-1,hyalurin/CD44, and the like. As will be recognized by one skilled in theart, the foregoing examples are not exclusive.

Nucleic acids in chlorotoxin agents of the present invention may haveany of a variety of activities including, for example, as anti-cancer orother therapeutic agents, probes, primers, etc. Nucleic acids inchlorotoxin agents of the present invention may have enzymatic activity(e.g., ribozyme activity), gene expression inhibitory activity (e.g., asantisense or siRNA agents, etc), and/or other activities. Nucleic acidsin chlorotoxin agents of the present invention may be active themselvesor may be vectors that deliver active nucleic acid agents (e.g., throughreplication and/or transcription of a delivered nucleic acid). Forpurposes of the present specification, such vector nucleic acids areconsidered “therapeutic agents” if they encode or otherwise deliver atherapeutically active agent, even if they do not themselves havetherapeutic activity.

In certain embodiments, a chlorotoxin agent comprises a nucleic acidtherapeutic agent that comprises or encodes an antisense compound. Theterms “antisense compound or agent”, “antisense oligomer”, “antisenseoligonucleotide”, and “antisense oligonucleotide analog” are used hereininterchangeably, and refer to a sequence of nucleotide bases and asubunit-to-subunit backbone that allows the antisense compound tohybridize to a target sequence in an RNA by Watson-Crick base pairing toform an RNA oligomer heteroduplex within the target sequence. Theoligomer may have exact sequence complementarity within the targetsequence or near complementarity. Such antisense oligomers may block orinhibit translation of the mRNA containing the target sequence, orinhibit gene transcription. Antisense oligomers may bind todouble-stranded or single-stranded sequences.

Examples of antisense oligonucleotides suitable for use in the practiceof the present invention include, for example, those mentioned in thefollowing reviews: R. A Stahel et al., Lung Cancer, 2003, 41: S81-S88;K. F. Pirollo et al., Pharmacol. Ther., 2003, 99: 55-77; A. C. Stephensand R. P. Rivers, Curr. Opin. Mol. Ther., 2003, 5: 118-122; N. M. Deanand C. F. Bennett, Oncogene, 2003, 22: 9087-9096; N. Schiavone et al.,Curr. Pharm. Des., 2004, 10: 769-784; L. Vidal et al., Eur. J. Cancer,2005, 41: 2812-2818; T. Aboul-Fadl, Curr. Med. Chem., 2005, 12:2193-2214; M. E. Gleave and B. P. Monia, Nat. Rev. Cancer, 2005, 5:468-479; Y. S. Cho-Chung, Curr. Pharm. Des., 2005, 11: 2811-2823; E.Rayburn et al., Lett. Drug Design &Discov., 2005, 2: 1-18; E. R. Rayburnet al., Expert Opin. Emerg. Drugs, 2006, 11: 337-352; I. Tamm and M.Wagner, Mol. Biotechnol., 2006, 33: 221-238 (each of which isincorporated herein by reference in its entirety).

Examples of suitable antisense oligonucleotides include, for exampleolimerson sodium (also known as Genasense™ or G31239, developed byGenta, Inc., Berkeley Heights, N.J.), a phosphorothioate oligomertargeted towards the initiation codon region of the bcl-2 mRNA, which isa potent inhibitor of apoptosis and is overexpressed in many cancerincluding, follicular lymphomas, breast, colon and prostate cancers, andintermediate/high-grade lymphomas (C. A. Stein et al., Semin. Oncol.,2005, 32: 563-573; S. R. Frankel, Semin. Oncol., 2003, 30: 300-304).Other suitable antisense oligonucleotides include GEM-231 (HYB0165,Hybridon, Inc., Cambridge, Mass.), which is a mixed backboneoligonucleotide directed against cAMP-dependent protein kinase A (PKA)(S. Goel et al., Clin. Cancer Res., 203, 9: 4069-4076); Affinitak (ISIS3521 or aprinocarsen, ISIS pharmaceuticals, Inc., Carlsbad, Calif.), anantisense inhibitor of PKC-alpha; OGX-011 (Isis 112989, IsisPharmaceuticals, Inc.), a 2′-methoxyethyl modified antisenseoligonucleotide against clusterin, a glycoprotein implicated in theregulation of the cell cycle, tissue remodeling, lipid transport andcell death and which is overexpressed in cancers of breast, prostate andcolon; ISIS 5132 (Isis 112989, Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide complementary to a sequence of the3′-unstranslated region of the c-raf-1 mRNA (S. P. Henry et al.,Anticancer Drug Des., 1997, 12: 409-420; B. P. Monia et al., Proc. Natl.Acad. Sci. USA, 1996, 93: 15481-15484; C. M. Rudin et al., Clin. CancerRes., 2001, 7: 1214-1220); ISIS 2503 (Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide antisense inhibitor of human H-ras mRNAexpression (J. Kurreck, Eur. J. Biochem., 2003, 270: 1628-1644);oligonucleotides targeting the X-linked inhibitor of apoptosis protein(XIAP), which blocks a substantial portion of the apoptosis pathway,such as GEM 640 (AEG 35156, Aegera Therapeutics Inc. and Hybridon, Inc.)or targeting survivin, an inhibitor of apoptosis protein (IAP), such asISIS 23722 (Isis Pharmaceuticals, Inc.), a 2′-O-methoxyethyl chimericoligonucleotide; MG98, which targets DNA methyl transferase; andGTI-2040 (Lorus Therapeutics, Inc. Toronto, Canada), a 20-meroligonucleotide that is complementary to a coding region in the mRNA ofthe R2 small subunit component of human ribonucleotide reductase.

Other suitable antisense oligonucleotides include antisenseoligonucleotides that are being developed against Her-2/neu, c-Myb,c-Myc, and c-Raf (see, for example, A. Biroccio et al., Oncogene, 2003,22: 6579-6588; Y. Lee et al., Cancer Res., 2003, 63: 2802-2811; B. Lu etal., Cancer Res., 2004, 64: 2840-2845; K. F. Pirollo et al., Pharmacol.Ther., 2003, 99: 55-77; and A. Rait et al., Ann. N.Y. Acad. Sci., 2003,1002: 78-89).

In certain embodiments, anchlorotoxin agent of the present inventioncomprises a nucleic acid anti-cancer agent that comprises or encodes aninterfering RNA molecule. The terms “interfering RNA” and “interferingRNA molecule” are used herein interchangeably, and refer to an RNAmolecule that can inhibit or downregulate gene expression or silence agene in a sequence-specific manner, for example by mediating RNAinterference (RNAi). RNA interference (RNAi) is an evolutionarilyconserved, sequence-specific mechanism triggered by double-stranded RNA(dsRNA) that induces degradation of complementary target single-strandedmRNA and “silencing” of the corresponding translated sequences (McManusand Sharp, 2002, Nature Rev. Genet., 2002, 3: 737). RNAi functions byenzymatic cleavage of longer dsRNA strands into biologically active“short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides inlength (Elbashir et al., Genes Dev., 2001, 15: 188). RNA interferencehas emerged as a promising approach for therapy of cancer.

An interfering RNA suitable for use in the practice of the presentinvention can be provided in any of several forms. For example, aninterfering RNA can be provided as one or more of an isolated shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),or short hairpin RNA (shRNA).

Examples of interfering RNA molecules suitable for use in the presentinvention include, for example, the iRNAs cited in the followingreviews: O. Milhavet et al., Pharmacol. Rev., 2003, 55: 629-648; F. Biet al., Curr. Gene. Ther., 2003, 3: 411-417; P. Y. Lu et al., Curr.Opin. Mol. Ther., 2003, 5: 225-234; I. Friedrich et al., Semin. CancerBiol., 2004, 14: 223-230; M. Izquierdo, Cancer Gene Ther., 2005, 12:217-227; P. Y. Lu et al., Adv. Genet., 2005, 54: 117-142; G. R. Devi,Cancer Gene Ther., 2006, 13: 819-829; M. A. Behlke, Mol. Ther., 2006,13: 644-670; and L. N. Putral et al., Drug News Perspect., 2006, 19:317-324 (each of which is incorporated herein by reference in itsentirety).

Other examples of suitable interfering RNA molecules include, but arenot limited to, p53 interfering RNAs (e.g., T. R. Brummelkamp et al.,Science, 2002, 296: 550-553; M. T. Hemman et al., Nat. Genet., 2003, 33:396-400); interfering RNAs that target the bcr-abl fusion, which isassociated with development of chronic myeloid leukemia and acutelymphoblastic leukemia (e.g., M. Scherr et al., Blood, 2003, 101:1566-1569; M. J. Li et al., Oligonucleotides, 2003, 13: 401-409),interfering RNAs that inhibit expression of NPM-ALK, a protein that isfound in 75% of anaplastic large cell lymphomas and leads to expressionof a constitutively active kinase associated with tumor formation (U.Ritter et al., Oligonucleotides, 2003, 13: 365-373); interfering RNAsthat target oncogenes, such as Raf-1 (T. F. Lou et al.,Oligonucleotides, 2003, 13: 313-324), K-Ras (T. R. Brummelkamp et al.,Cancer Cell, 2002, 2: 243-247), erbB-2 (G. Yang et al., J. Biol. Chem.,2004, 279: 4339-4345); interfering RNAs that target b-catenin protein,whose over-expression leads to transactivation of the T-cell factortarget genes, which is thought to be the main transforming event incolorectal cancer (M. van de Wetering et al., EMBO Rep., 2003, 4:609-615).

C. Labeling Moieties

In certain embodiments, a chlorotoxin agent is labeled with at least onelabeling moiety. For example, one or more chlorotoxin moieties and/orone or more therapeutic moieties within a chlorotoxin agent may belabeled with a labeling moiety.

The role of a labeling moiety is to facilitate detection of thechlorotoxin agent after binding to the tissue to be tested. Preferably,the labeling moiety is selected such that it generates a signal that canbe measured and whose intensity is related to (e.g., proportional to)the amount of diagnostic agent bound to the tissue.

Preferably, labeling does not substantially interfere with the desiredbiological or pharmaceutical activity of the chlorotoxin agent. Incertain embodiments, labeling involves attachment or incorporation ofone or more labeling moieties to a chlorotoxin moiety, preferably tonon-interfering positions on the peptide sequence of the chlorotoxinmoiety. Such non-interfering positions are positions that do notparticipate in the specific binding of the chlorotoxin moiety to tumorcells.

A labeling moiety may be any entity that allows detection ofachlorotoxin agent after binding to a tissue or system of interest. Anyof a wide variety of detectable agents can be used as labeling moietiesin chlorotoxin agents of the present invention. A labeling moiety may bedirectly detectable or indirectly detectable. Examples of labelingmoieties include, but are not limited to: various ligands, radionuclides(e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re,¹¹¹In, ⁹⁰Y, ^(99m)Tc, ¹⁷⁷Lu, etc.), fluorescent dyes (for specificexemplary fluorescent dyes, see below), chemiluminescent agents (suchas, for example, acridinum esters, stabilized dioxetanes, and the like),bioluminescent agents, spectrally resolvable inorganic fluorescentsemiconductors nanocrystals (i.e., quantum dots), metal nanoparticles(e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagneticmetal ions, enzymes (for specific examples of enzymes, see below),colorimetric labels (such as, for example, dyes, colloidal gold, and thelike), biotin, dioxigenin, haptens, and proteins for which antisera ormonoclonal antibodies are available.

In certain embodiments, a labeling moiety comprises a fluorescent label.Numerous known fluorescent labeling moieties of a wide variety ofchemical structures and physical characteristics are suitable for use inthe practice of methods of diagnosis of the present invention. Suitablefluorescent dyes include, but are not limited to, fluorescein andfluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.),Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes(e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™, etc.), Alexa etc.) Fluor dyes(e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660,Alexa Fluor 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPYTMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc.), IRDyes(e.g., IRD40, IRD 700, IRD 800, etc.), and the like. For more examplesof suitable fluorescent dyes and methods for coupling fluorescent dyesto other chemical entities such as proteins and peptides, see, forexample, “The Handbook of Fluorescent Probes and Research Products”,9^(th) Ed., Molecular Probes, Inc., Eugene, Oreg. Favorable propertiesof fluorescent labeling agents include high molar absorptioncoefficient, high fluorescence quantum yield, and photostability. Incertain embodiments, labeling fluorophores desirably exhibit absorptionand emission wavelengths in the visible (i.e., between 400 and 750 nm)rather than in the ultraviolet range of the spectrum (i.e., lower than400 nm).

In certain embodiments, a labeling moiety comprises an enzyme. Examplesof suitable enzymes include, but are not limited to, those used in anELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase, etc. Other examples include beta-glucuronidase,beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may beconjugated to a chlorotoxin moiety using a linker group such as acarbodiimide, a diisocyanate, a glutaraldehyde, and the like.

In certain embodiments, a labeling moiety comprises a radioisotope thatis detectable by Single Photon Emission Computed Tomography (SPECT) orPosition Emission Tomography (PET). Examples of such radionuclidesinclude, but are not limited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), astatine-221 (²¹¹At),copper-67 (⁶⁷Cu), copper-64 (⁶⁴Cu), rhenium-186 (¹⁸⁶Re), rhenium-186(¹⁸⁸Re), phosphorus-32 (³²P), samarium-153 (¹⁵³Sm), lutetium-177(¹¹⁷Lu), technetium-99m (^(99m)Tc), gallium-67 (⁶⁷Ga), indium-111(¹¹¹In), and thallium-201 (²⁰¹Tl).

In certain embodiments, a labeling moiety comprises a radioisotope thatis detectable by Gamma camera. Examples of such radioisotopes include,but are not limited to, iodine-131 (¹³¹I), and technetium-99m(^(99m)Tc).

In certain embodiments, a labeling moiety comprises a paramagnetic metalion that is a good contrast enhancer in Magnetic Resonance Imaging(MRI). Examples of such paramagnetic metal ions include, but are notlimited to, gadolinium III (Gd³⁺), chromium III (Cr³⁺), dysprosium III(Dy³⁺), iron III (Fe³⁺), manganese II (Mn²⁺), and ytterbium III (Yb³⁺).In certain embodiments, the labeling moieties comprises gadolinium III(Gd³⁺). Gadolinium is an FDA-approved contrast agent for MRI, whichaccumulates in abnormal tissues causing these abnormal areas to becomevery bright (enhanced) on the magnetic resonance image. Gadolinium isknown to provide great contrast between normal and abnormal tissues indifferent areas of the body, in particular in the brain.

In certain embodiments, a labeling moiety comprises a stableparamagnetic isotope detectable by nuclear magnetic resonancespectroscopy (MRS). Examples of suitable stable paramagnetic isotopesinclude, but are not limited to, carbon-13 (¹³C) and fluorine-19 (¹⁹F).

D. Formation of Chlorotoxin Agents

In certain embodiments, a chlorotoxin agent comprises at least onechlorotoxin moiety associated with at least one therapeutic moiety.Thus, a chlorotoxin agent results from the association (e.g., binding,interaction, fusion, or coupling) of at least two other molecules.

Association between a chlorotoxin moiety and a therapeutic moiety withina chlorotoxin agent may be covalent or non-covalent. Irrespective of thenature of the binding, interaction, or coupling, the association betweena chlorotoxin moiety and a therapeutic moiety is preferably selective,specific and strong enough so that the chlorotoxin agent does notdissociate before or during transport/delivery to and into the tumor.Association between a chlorotoxin moiety and a therapeutic moiety withina chlorotoxin agent may be achieved using any chemical, biochemical,enzymatic, or genetic coupling known to one skilled in the art.

In certain embodiments, association between a chlorotoxin moiety and atherapeutic moiety is non-covalent. Examples of non-covalentinteractions include, but are not limited to, hydrophobic interactions,electrostatic interactions, dipole interactions, van der Waalsinteractions, and hydrogen bonding.

In certain embodiments, association between achlorotoxin moiety and atherapeutic moiety is covalent. As will be appreciated by one skilled inthe art, the moieties may be attached to each other either directly orindirectly (e.g., through a linker, as described below).

In certain embodiments, the chlorotoxin moiety and therapeutic moietyare directly covalently linked to each other. Direct covalent bindingcan be through a linkage such as an amide, ester, carbon-carbon,disulfide, carbamate, ether, thioether, urea, amine, or carbonatelinkage. The covalent binding can be achieved by taking advantage offunctional groups present on the chlorotoxin moiety and/or thetherapeutic moiety. Alternatively, a non-critical amino acid may bereplaced by another amino acid that will introduce a useful group(amino, carboxy or sulfhydryl) for coupling purposes. Alternatively, anadditional amino acid may be added to the chlorotoxin moiety tointroduce a useful group (amino, carboxy or sulfhydryl) for couplingpurposes. Suitable functional groups that can be used to attach moietiestogether include, but are not limited to, amines, anhydrides, hydroxylgroups, carboxy groups, thiols, and the like. An activating agent, suchas a carbodiimide, can be used to form a direct linkage. A wide varietyof activating agents are known in the art and are suitable for linking atherapeutic agent and a chlorotoxin moiety.

In other embodiments, a chlorotoxin moiety and a therapeutic moietywithin a chlorotoxin agent are indirectly covalently linked to eachother via a linker group. This can be accomplished by using any numberof stable bifunctional agents well known in the art, includinghomofunctional and heterofunctional agents (for examples of such agents,see, e.g., Pierce Catalog and Handbook). The use of a bifunctionallinker differs from the use of an activating agent in that the formerresults in a linking moiety being present in the resulting chlorotoxinagent, whereas the latter results in a direct coupling between the twomoieties involved in the reaction. The role of a bifunctional linker maybe to allow reaction between two otherwise inert moieties. Alternativelyor additionally, the bifunctional linker that becomes part of thereaction product may be selected such that it confers some degree ofconformational flexibility to the chlorotoxin agent (e.g., thebifunctional linker comprises a straight alkyl chain containing severalatoms, for example, the straight alkyl chain contains between 2 and 10carbon atoms). Alternatively or additionally, the bifunctional linkermay be selected such that the linkage formed between a chlorotoxinmoiety and therapeutic moiety is cleavable, e.g. hydrolysable (forexamples of such linkers, see e.g. U.S. Pat. Nos. 5,773,001; 5,739,116and 5,877,296, each of which is incorporated herein by reference in itsentirety). Such linkers are for example preferably used when higheractivity of the chlorotoxin moiety and/or of the therapeutic moiety isobserved after hydrolysis of the conjugate. Exemplary mechanisms bywhich a therapeutic moiety may be cleaved from a chlorotoxin moietyinclude hydrolysis in the acidic pH of the lysosomes (hydrazones,acetals, and cis-aconitate-like amides), peptide cleavage by lysosomalenzymes (the capthepsins and other lysosomal enzymes), and reduction ofdisulfides). Another mechanism by which a therapeutic moiety is cleavedfrom the chlorotoxin agent includes hydrolysis at physiological pHextra- or intra-cellularly. This mechanism applies when the crosslinkerused to couple the therapeutic moiety to the chlorotoxin moiety is abiodegradable/bioerodible entity, such as polydextran and the like.

For example, hydrazone-containing chlorotoxin agents can be made withintroduced carbonyl groups that provide the desired release properties.Chlorotoxin agents can also be made with a linker that comprise an alkylchain with a disulfide group at one end and a hydrazine derivative atthe other end. Linkers containing functional groups other thanhydrazones also have the potential to be cleaved in the acidic milieu oflysosomes. For example, chlorotoxin agents can be made fromthiol-reactive linkers that contain a group other than a hydrazone thatis cleavable intracellularly, such as esters, amides, andacetals/ketals.

Another example of class of pH sensitive linkers are the cis-aconitates,which have a carboxylic acid group juxtaposed to an amide group. Thecarboxylic acid accelerates amide hydrolysis in the acidic lysosomes.Linkers that achieve a similar type of hydrolysis rate acceleration withseveral other types of structures can also be used.

Another potential release method for chlorotoxin agents is the enzymatichydrolysis of peptides by the lysosomal enzymes. In one example, apeptidic toxin is attached via an amide bond to para-aminobenzyl alcoholand then a carbamate or carbonate is made between the benzyl alcohol andthe therapeutic moiety. Cleavage of the peptide leads to collapse of theamino benzyl carbamate or carbonate, and release of the therapeuticmoiety. In another example, a phenol can be cleaved by collapse of thelinker instead of the carbamate. In another variation, disulfidereduction is used to initiate the collapse of a para-mercaptobenzylcarbamate or carbonate.

In embodiments where a therapeutic moiety within a chlorotoxin agent isa protein, a polypeptide or a peptide, the chlorotoxin agent may be afusion protein. As already defined above, a fusion protein is a moleculecomprising two or more proteins or peptides linked by a covalent bondvia their individual peptide backbones. Fusion proteins used in methodsof the present invention can be produced by any suitable method known inthe art. For example, they can be produced by direct protein syntheticmethods using a polypeptide synthesizer. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments that can subsequently be annealed and re-amplified to generatea chimeric gene sequence. Fusion proteins can be obtained by standardrecombinant methods (see, for example, Maniatis et al. “MolecularCloning: A Laboratory Manual”, 2^(nd) Ed., 1989, Cold Spring HarborLaboratory, Cold Spring, N.Y.). These methods generally comprise (1)construction of a nucleic acid molecule that encodes the desired fusionprotein; (2) insertion of the nucleic acid molecule into a recombinantexpression vector; (3) transformation of a suitable host cell with theexpression vector; and (4) expression of the fusion protein in the hostcell. Fusion proteins produced by such methods may be recovered andisolated, either directly from the culture medium or by lysis of thecells, as known in the art. Many methods for purifying proteins producedby transformed host cells are well-known in the art. These include, butare not limited to, precipitation, centrifugation, gel filtration, and(ion-exchange, reverse-phase, and affinity) column chromatography. Otherpurification methods have been described (see, for example, Deutscher etal. “Guide to Protein Purification” in Methods in Enzymology, 1990, Vol.182, Academic Press).

As can readily be appreciated by one skilled in the art, a chlorotoxinagent used in methods of the present invention can comprise any numberof chlorotoxin moieties and any number of therapeutic moieties,associated to one another by any number of different ways. The design ofa conjugate will be influenced by its intended purpose(s) and theproperties that are desirable in the particular context of its use.Selection of a method to associate or bind a chlorotoxin moiety to atherapeutic moiety to form a chlorotoxin agent is within the knowledgeof one skilled in the art and will generally depend on the nature of theinteraction desired between the moieties (i.e., covalent vs.non-covalent and/or cleavable vs. non-cleavable), the nature of thetherapeutic moiety, the presence and nature of functional chemicalgroups on the moieties involved and the like.

In labeled chlorotoxin agents, association between a chlorotoxin moiety(or therapeutic moiety) and a labeling moiety may be covalent ornon-covalent. In case of covalent association, the chlorotoxin (ortherapeutic) and labeling moieties may be attached to each other eitherdirectly or indirectly, as described above.

In certain embodiments, association between a chlorotoxin moiety (ortherapeutic moiety) and a labeling moiety is non-covalent. Examples ofnon-covalent associations include, but are not limited to, hydrophobicinteractions, electrostatic interactions, dipole interactions, van derWaals interactions, and hydrogen bonding. For example, a labeling moietycan be non-covalently attached to a chlorotoxin moiety (or therapeuticmoiety) by chelation (e.g., a metal isotope can be chelated to a polyHisregion attached, e.g., fused, to a chlorotoxin moiety).

In certain embodiments, a chlorotoxin moiety (or therapeutic moiety) isisotopically labeled (i.e., it contains one or more atoms that have beenreplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number usually found in nature). Alternativelyor additionally, an isotope may be attached to a chlorotoxin moietyand/or therapeutic moiety.

As can readily be appreciated by one skilled in the art, a labeledchlorotoxin agent used in certain methods of the present invention cancomprise any number of chlorotoxin moieties, any number of therapeuticmoieties, and any number of labeling moieties, associated to one anotherby any number of different ways. The design of a labeled chlorotoxinagent will be influenced by its intended purpose(s), the properties thatare desirable in the context of its use, and the method selected fromthe detection.

E. Modifications

In certain embodiments, chlorotoxin agents are modified by covalentattachment to macromolecules such as polymers. Without wishing to bebound by any particular theory, covalent attachment of, for example,polymers, may mask the chlorotoxin agent antigenically such that theagent's bioavailability and/or tolerance in an animal's body isimproved. An example of such a polymer is polyethylene glycol (PEG),which can often be covalently attached to N and/or C termini and/or tocysteines in peptides and/or polypeptides. “PEGylation” refers to thecovalent addition of PEG to a molecule. In some embodiments, chlorotoxinagents are not modified at any site. In some embodiments, chlorotoxinagents are modified (e.g., by PEGylation) at one site per molecule. Insome embodiments, chlorotoxin agents are modified (e.g., by PEGylation)at more than one site per molecule.

In some embodiments, such modifications increase the half life ofchlorotoxin agents in vivo. For example, the half life may be at leastapproximately 10 hours, at least approximately 16 hours, etc. (See,e.g., Example 9). Such improved bioavailability may in some embodimentsfacilitate dosing regimens that involve lower frequencies of dosing.(Dosing regimens are discussed herein.)

II. Methods of Treatment and/or Detection

Methods of treatment of the invention include administration of aneffective dose of a chlorotoxin agent, or a pharmaceutical compositionthereof, to an individual in need thereof (e.g., a individual who has,has had, is at risk of developing, and/or is susceptible to at least onetumor metastastis). Thus, methods of treatment of the present inventionmay be used for reducing the sizes and/or numbers of tumor metastases,inhibiting the growth and/or formation of metastases, and/or prolongingthe survival time of mammals (including humans) suffering frommetastatic cancers and metastatic cancer conditions.

Without wishing to be bound by any particular theory, we note thatformation of new blood vessels (angiogenesis) may be important fordeveloping and/or maintaining metastases. As demonstrated in Example 4,chlorotoxin can inhibit new blood vessel formation. Chlorotoxin may alsocause the regression of newly developed blood vessels (neovasculature).In some embodiments of the invention, neovasculature of at least onemetastasis regresses. In some embodiments of the invention,neovascularization is inhibited.

A. Indications

Throughout the specification, the convention of naming a metastastictumor after the site of its primary origin is used. Thus, for example,“metastatic prostate cancer” refers to cancer originating from theprostate that has spread to other organs, regardless of the location ofthe metastasis. Metastases may form in a variety of organs including,for example, brain, lung, bone, liver, lymph nodes, ovary, etc. Certainkinds of tumors may typically metastasize to certain organs. Forexample, melanomas often metastasize to the brain, prostate cancersoften metastasize to bone, stomach cancers in women often metastasize tothe ovary, breast cancers often metastasize to bone, and colon cancersoften metastasize to the liver. Inventive methods may be used to treatmetastases in a variety of organs as described above, includingmetastases that are distant from the site of the primary tumor.Furthermore, because chlorotoxin agents may cross the blood/brainbarrier (see, for example, Examples 2 and 3) inventive methods may beused to treat metastases in brain.

Primary tumors may often spread to nearby lymph nodes. Inventive methodsmay be used to control or eliminate spreading to lymph nodes as well.Cancer/tumor cells may break away from a primary tumor and metastasizeby traveling through the bloodstream or lymphatic channels. In certainembodiments of the invention, for example some of those in which thechlorotoxin agent is systemically delivered, such metastasizing cellsare bound by the chlorotoxin agent and targeted for destruction.

In some embodiments, inventive treatment methods further comprisedetecting at least one metastasis prior to administration of thechlorotoxin agent. In some such embodiments, detecting at least onemetastasis comprises administering an effective dose of a labeledchlorotoxin agent.

It will be understood, nevertheless, that inventive methods can be usedto treat individuals having, having had, or at risk of having one ormore metastases even though the location or existence of the one or moremetastases may not be known. A patient may not have been diagnosed ashaving any metastases at all, or only a subset of metastases in thepatient may have been identified and/or located. In some embodiments ofinvention, the chlorotoxin agent is delivered systemically, resulting inchlorotoxin agent being delivered throughout the body. Thus, it is notnecessary to target delivery of chlorotoxin agent to a particular tissueor set of tissues in order to effect delivery of chlorotoxin agent tometastases.

Examples of primary cancers and cancer conditions that can develop intometastastic cancers that can be treated according to the presentinvention include, but are not limited to, tumors of the brain andcentral nervous system (e.g., tumors of the meninges, brain, spinalcord, cranial nerves and other parts of the CNS, such as glioblastomasor medulloblastomas); head and/or neck cancer, breast tumors, tumors ofthe circulatory system (e.g., heart, mediastinum and pleura, and otherintrathoracic organs, vascular tumors, and tumor-associated vasculartissue); tumors of the blood and lymphatic system (e.g., Hodgkin'sdisease, Non-Hodgkin's disease lymphoma, Burkitt's lymphoma,AIDS-related lymphomas, malignant immunoproliferative diseases, multiplemyeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloidleukemia, acute or chronic lymphocytic leukemia, monocytic leukemia,other leukemias of specific cell type, leukemia of unspecified celltype, unspecified malignant neoplasms of lymphoid, haematopoietic andrelated tissues, such as diffuse large cell lymphoma, T-cell lymphoma orcutaneous T-cell lymphoma); tumors of the excretory system (e.g.,kidney, renal pelvis, ureter, bladder, and other urinary organs); tumorsof the gastrointestinal tract (e.g., oesophagus, stomach, smallintestine, colon, colorectal, rectosigmoid junction, rectum, anus, andanal canal); tumors involving the liver and intrahepatic bile ducts,gall bladder, and other parts of the biliary tract, pancreas, and otherdigestive organs; tumors of the oral cavity (e.g., lip, tongue, gum,floor of mouth, palate, parotid gland, salivary glands, tonsil,oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites ofthe oral cavity); tumors of the reproductive system (e.g., vulva,vagina, Cervix uteri, uterus, ovary, and other sites associated withfemale genital organs, placenta, penis, prostate, testis, and othersites associated with male genital organs); tumors of the respiratorytract (e.g., nasal cavity, middle ear, accessory sinuses, larynx,trachea, bronchus and lung, such as small cell lung cancer and non-smallcell lung cancer); tumors of the skeletal system (e.g., bone andarticular cartilage of limbs, bone articular cartilage and other sites);tumors of the skin (e.g., malignant melanoma of the skin, non-melanomaskin cancer, basal cell carcinoma of skin, squamous cell carcinoma ofskin, mesothelioma, Kaposi's sarcoma); and tumors involving othertissues including peripheral nerves and autonomic nervous system,connective and soft tissue, retroperitoneoum and peritoneum, eye andadnexa, thyroid, adrenal gland, and other endocrine glands and relatedstructures, secondary and unspecified malignant neoplasms of lymphnodes, secondary malignant neoplasm of respiratory and digestive systemsand secondary malignant neoplasms of other sites.

In certain embodiments of the present invention, inventive compositionsand methods are used in the treatment of metastatic sarcomas. In someembodiments, compositions and methods of the present invention are usedin the treatment of metastatic cancers originating from bladder cancer,breast cancer, chronic lymphoma leukemia, head and neck cancer,endometrial cancer, Non-Hodgkin's lymphoma, non-small cell lung cancer,ovarian cancer, pancreatic cancer, and prostate cancer.

In certain embodiments of the present invention, compositions andmethods are used for the treatment of metastatic tumors ofneuroectodermal origin. Any metastatic tumor of neuroectodermal originpresent in a human patient can generally be treated using acomposition/method of the present invention. In certain embodiments, themetastatic tumor of neuroectodermal origin affecting the patient is amember of the group consisting of gliomas, meningiomas, ependymomas,medulloblastomas, neuroblastomas, gangliomas, pheochromocytomas,melanomas, peripheral primitive neuroectodermal tumors, small cellcarcinoma of the lung, Ewing's sarcoma, and metastatic tumors ofneuroectodermal origin in the brain. In some embodiments, the metastatictumor of neuroectodermal origin is melanoma. In some such embodiments,the melanoma is cutaneous or intraocular melanoma.

In certain embodiments, the metastatic tumor of neuroectodermal originaffects the brain of the patient. In certain embodiments, the braintumor is a glioma. About half of all primary brain tumors are gliomas.There are 4 main types of glioma: astrocytoma (which is the most commontype of glioma in both adults and children), ependymoma,oligodendroglioma, and mixed glioma. Gliomas can be classified accordingto their location: infratentorial (i.e., located in the lower part ofthe brain, found mostly in children patients) or supratentorial (i.e.,located in the upper part of the brain, found mostly in adult patients).

Gliomas are further categorized according to their grade, which isdetermined by pathologic evaluation of the tumor. The World HealthOrganization (WHO) has developed a grading system, from Grade I gliomas,which tend to be the least aggressive, to Grade IV gliomas, which tendto be the most aggressive and malignant. Examples of low grade (i.e.,Grade I or Grade II) gliomas include, but are not limited to, pilocyticastrocytoma (also called juvenile pilocytic astrocytoma), fibrillaryastrocytoma, pleomorphic xantroastrocytomoa, and desembryoplasticneuroepithelial tumor. High-grade gliomas encompass Grade III gliomas(e.g., anaplastic astrocytoma, AA) and Grade IV gliomas (glioblastomamultiforme, GBM). Anaplastic astrocytoma is most frequent among men andwomen in theirs 30s-50s, and accounts for 4% of all brain tumors.Glioblastoma multiforme, the most invasive type of glial tumor, is mostcommon in men and women in their 50s-70s and accounts for 23% of allprimary brain tumors. The prognosis is the worst for Grade IV gliomas,with an average survival time of 12 months. In certain embodiments,methods of the present invention are used for the treatment ofhigh-grade gliomas.

Despite aggressive treatment, gliomas usually recur, often with a highergrade and sometimes with a different morphology. While recurrencevaries, Grade IV gliomas invariably recur. Thus, in certain embodiments,methods of the present invention are used for the treatment ofmetastatic recurrent gliomas, in particular, recurrent high-gradegliomas.

Metastatic tumors that can be treated using compositions and methods ofthe present invention also include metastatic tumors that are refractoryto treatment with other chemotherapeutics. The term “refractory”, whenused herein in reference to a tumor means that the tumor (and/ormetastases thereof), upon treatment with at least one chemotherapeuticother than an inventive composition, shows no or only weakanti-proliferative response (i.e., no or only weak inhibition of tumorgrowth) after the treatment with such a chemotherapeutic agent—that is,a tumor that cannot be treated at all or only with unsatisfying resultswith other (preferably standard) chemotherapeutics. The presentinvention, where treatment of refractory tumors and the like ismentioned, is to be understood to encompass not only (i) tumors whereone or more chemotherapeutics have already failed during treatment of apatient, but also (ii) tumors that can be shown to be refractory byother means, e.g., biopsy and culture in the presence ofchemotherapeutics.

Patients who can receive a treatment according to the present inventiongenerally include any patient who is or has been diagnosed with a tumor.In some embodiments, the patient is diagnosed as having one or moremetastasis. In some embodiments, the patient is diagnosed as having atumor that is known to metastasize. In some embodiments, the patient isdiagnosed as having a tumor that is determined to be at a stage duringwhich metastasis is likely or possible. In some embodiments, the patienthas had a tumor, but no longer exhibits signs of having the primarytumor; in some such embodiments, the patient has metastases neverthelessthat may be treated with inventive methods. As will be recognized by oneskilled in the art, different methods of diagnosis may be performeddepending on the location and nature of the tumor and/or metastases,including imaging, biopsy, etc.

B. Dosages and Administrations

In a method of treatment of the present invention, a chlorotoxin agent,or a pharmaceutical composition thereof, will generally be administeredin such amounts and for such a time as is necessary or sufficient toachieve at least one desired result. For example, a chlorotoxin agentcan be administered in such amounts and for such a time that it killscancer cells, reduces tumor size, reduces the size of one or moremetastases, inhibits or delay tumor growth or metastasis, prolongs thesurvival time of patients, or otherwise yields clinical benefits.

A treatment according to the present invention may consist of a singledose or a plurality of doses over a period of time. Administration maybe one or multiple times daily, weekly (or at some other multiple dayinterval) or on an intermittent schedule. The exact amount of achlorotoxin agent, or pharmaceutical composition thereof, to beadministered will vary from subject to subject and will depend onseveral factors (see below).

Chlorotoxin agents, or pharmaceutical compositions thereof, may beadministered using any administration route effective for achieving thedesired therapeutic effect. In certain embodiments of the invention,chlorotoxin agents (or pharmaceutical compositions thereof) aredelivered systemically. Typical systemic routes of administrationinclude, but are not limited to, intramuscular, intravenous, pulmonary,and oral routes. Systemic administration may also be performed, forexample, by infusion or bolus injection, or by absorption throughepithelial or mucocutaneous linings (e.g., oral, mucosa, rectal andintestinal mucosa, etc). In certain embodiments, the chlorotoxin agentis administered intravenously. Exemplary procedures for the intravenousadministration of a chlorotoxin agent in human patients are described inExample 2.

Depending on the route of administration, effective doses may becalculated according to the body weight; body surface area; primaryorgan/tumor size; and/or number, sizes, and/or types of metastases ofthe subject to be treated. Optimization of the appropriate dosages canreadily be made by one skilled in the art in light of pharmacokineticdata observed in human clinical trials. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of the drugs, e.g., the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anypresent infection, time of administration, the use (or not) of othertherapies, and other clinical factors. As studies are conducted usingchlorotoxin agents, further information will emerge regarding theappropriate dosage levels and duration of treatment.

Typical dosages comprise 1.0 pg/kg body weight to 100 mg/kg body weight.For example, for systemic administration, dosages may be 100.0 ng/kgbody weight to 10.0 mg/kg body weight.

More specifically, in certain embodiments where a chlorotoxin agent isadministered intravenously, dosing of the agent may compriseadministration of one or more doses comprising about 0.005 mg/kg toabout 5 mg/kg, e.g., from about 0.005 mg/kg to about 5 mg/kg, from about0.01 mg/kg to about 4 mg/kg, from about 0.02 mg/kg to about 3 mg/kg,from about 0.03 mg/kg to about 2 mg/kg or from about 0.03 mg/kg to about1.5 mg/kg of chlorotoxin. For example, in certain embodiments, one ormore doses of chlorotoxin agent may be administered that each containsabout 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg,about 0.07 mg/kg, about 0.09 mg/kg, about 1.0 mg/kg or more than 1.0mg/kg of chlorotoxin. In other embodiments, one or more doses ofchlorotoxin agent may be administered that each contains about 0.05mg/kg, about 0.10 mg/kg, about 0.15 mg/kg, about 0.20 mg/kg, about 0.25mg/kg, about 0.30 mg/kg, about 0.35 mg/kg, about 0.40 mg/kg, about 0.45mg/kg, about 0.50 mg/kg, about 0.55 mg/kg, about 0.60 mg/kg, about 0.65mg/kg, about 0.70 mg/kg, about 0.75 mg/kg, about 0.80 mg/kg, about 0.85mg/kg, about 0.90 mg/kg, about 0.95 mg/kg, about 1.0 mg/kg, or more thanabout 1 mg/kg of chlorotoxin. In yet other embodiments, one or moredoses of chlorotoxin agent may be administered that each contains about1.0 mg/kg, about 1.05 mg/kg, about 1.10 mg/kg, about 1.15 mg/kg, about1.20 mg/kg, about 1.25 mg/kg, about 1.3 mg/kg, about 1.35 mg/kg, about1.40 mg/kg, about 1.45 mg/kg, about 1.50 mg/kg, or more than about 1.50mg/kg of chlorotoxin. In such embodiments, at treatment may compriseadministration of a single dose of chlorotoxin agent or administrationof 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or more than 6 doses. Twoconsecutive doses may be administered at 1 day interval, 2 daysinterval, 3 days interval, 4 days interval, 5 days interval, 6 daysinterval, 7 days interval, or more than 7 days interval (e.g., 10 days,2 weeks, or more than 2 weeks).

C. Combination Therapies

It will be appreciated that methods of treatment of the presentinvention can be employed in combination with additional therapies(i.e., a treatment according to the present invention can beadministered concurrently with, prior to, or subsequently to one or moredesired therapeutics or medical procedures). The particular combinationof therapies (therapeutics or procedures) to employ in such acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved.

For example, methods of treatment of the present invention can beemployed together with other procedures including surgery, radiotherapy(e.g., γ-radiation, neuron beam radiotherapy, electron beamradiotherapy, proton therapy, brachytherapy, systemic radioactiveisotopes), endocrine therapy, hyperthermia, and cryotherapy, dependingon the tumor to be treated.

In many cases of metastatic brain tumor, a treatment of the presentinvention will often be administered after surgery to remove the primarytumor. In the treatment of brain tumor, the main goal of surgery is toachieve a gross-total resection, i.e., removal of all visible primarytumor. One of the difficulties in achieving such a goal is that thesetumors are infiltrative, i.e., they tend to weave in and out amongnormal brain structures. Furthermore, there is a great variability inthe amount of tumor that can be safely removed from the brain of apatient. Removal is generally not possible if all or part of the tumoris located in a region of the brain controlling critical functions.Furthermore, it may not be possible or practical to remove and/ordestroy metastases at distant sites with surgery alone.

In many cases of metastatic brain tumor, a treatment of the presentinvention will often be administered in combination with (i.e.,concurrently with, prior to, or subsequently to) radiotherapy. Inconventional treatments, radiotherapy generally follows surgery.Radiation is generally given as a series of daily treatments (calledfractions) over several weeks. This “fractionated” approach toadministering radiation is important to maximize the destruction oftumor cells and minimize side effects on normal adjacent brain. The areaover which the radiation is administered (called the radiation field) iscarefully calculated to avoid including as much of normal brain as isfeasible.

Alternatively or additionally, methods of treatment of the presentinvention can be administered in combination with other therapeuticagents, such as agents that attenuate any adverse effects (e.g.,antiemetics, etc.) and/or with other approved chemotherapeutic drugs.Examples of chemotherapeutics include, but are not limited to,alkylating drugs (e.g., mechlorethamine, chlorambucil, cyclophosphamide,melphalan, ifosfamide, etc.), antimetabolites (e.g., methotrexate,etc.), purine antagonists and pyrimidine antagonists (e.g.,6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine, etc.),spindle poisons (e.g., vinblastine, vincristine, vinorelbine,paclitaxel, etc.), podophyllotoxins (e.g., etoposide, irinotecan,topotecan, etc.), antibiotics (e.g., doxorubicin, bleomycin, mitomycin,etc.), nitrosureas (e.g., carmustine, lomustine, nomustine, etc.),inorganic ions (e.g., cisplatin, carboplatin, etc.), enzymes (e.g.,asparaginase, etc.), and hormones (e.g., tamoxifen, leuprolide,flutamide, megestrol, etc.), to name a few. For a more comprehensivediscussion of updated cancer therapies see, http://www.cancer.gov/, alist of the FDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Methods of the present invention can also be employed together with oneor more further combinations of cytotoxic agents as part of a treatmentregimen, wherein the further combination of cytotoxic agents is selectedfrom: CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone, andprocarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone); COP (cyclophosphamide, vincristine, and prednisone);CAP-BOP (cyclophosphamide, doxorubicin, procarbazine, bleomycin,vincristine, and prednisone); m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone, andleucovorin); ProMACE-MOPP (prednisone, methotrexate, doxorubicin,cyclophosphamide, etoposide, leucovorin, mechloethamine, vincristine,prednisone, and procarbazine); ProMACE-CytaBOM (prednisone,methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin,cytarabine, bleomycin, and vincristine); MACOP-B (methotrexate,doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, andleucovorin); MOPP (mechloethamine, vincristine, prednisone, andprocarbazine); ABVD (adriamycin/doxorubicin, bleomycin, vinblastine, anddacarbazine); MOPP (mechloethamine, vincristine, prednisone andprocarbazine) alternating with ABV (adriamycin/doxorubicin, bleomycin,and vinblastine); MOPP (mechloethamine, vincristine, prednisone, andprocarbazine) alternating with ABVD (adriamycin/doxorubicin, bleomycin,vinblastine, and dacarbazine); ChlVPP (chlorambucil, vinblastine,procarbazine, and prednisone); IMVP-16 (ifosfamide, methotrexate, andetoposide); MIME (methyl-gag, ifosfamide, methotrexate, and etoposide);DHAP (dexamethasone, high-dose cytaribine, and cisplatin); ESHAP(etoposide, methylpredisolone, high-dose cytarabine, and cisplatin);CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, andbleomycin); CAMP (lomustine, mitoxantrone, cytarabine, and prednisone);CVP-1 (cyclophosphamide, vincristine, and prednisone), ESHOP (etoposide,methylpredisolone, high-dose cytarabine, vincristine and cisplatin);EPOCH (etoposide, vincristine, and doxorubicin for 96 hours with bolusdoses of cyclophosphamide and oral prednisone), ICE (ifosfamide,cyclophosphamide, and etoposide), CEPP(B) (cyclophosphamide, etoposide,procarbazine, prednisone, and bleomycin), CHOP-B (cyclophosphamide,doxorubicin, vincristine, prednisone, and bleomycin), CEPP-B(cyclophosphamide, etoposide, procarbazine, and bleomycin), and P/DOCE(epirubicin or doxorubicin, vincristine, cyclophosphamide, andprednisone).

As will be appreciated by one skilled in the art, the selection of oneor more therapeutic agents to be administered in combination with amethod of treatment of the present invention will depend on themetastatic tumor to be treated.

For example, chemotherapeutic drugs prescribed for brain tumors include,but are not limited to, temozolomide (Temodar®), procarbazine(Matulane®), and lomustine (CCNU), which are taken orally; vincristine(Oncovin® or Vincasar PFS®), cisplatin (Platinol®), carmustine (BCNU,BiCNU), and carboplatin (Paraplatin®), which are administeredintravenously; and mexotrexate (Rheumatrex® or Trexall®), which can beadministered orally, intravenously or intrathecally (i.e., injecteddirectly into spinal fluid). BCNU is also given under the form of apolymer wafer implant during surgery (Giadel® wafers). One of the mostcommonly prescribed combination therapy for brain tumors is PCV(procarbazine, CCNU, and vincristine) which is usually given every sixweeks.

In embodiments where the tumor to be treated is a brain tumor ofneuroectodermal origin, a method of the present invention may be used incombination with agents for the management of symptoms such as seizuresand cerebral edema. Examples of anticonvulsants successfullyadministered to control seizures associated with brain tumors include,but are not limited to, phenyloin (Dilantin®), Carbamazepine (Tegretol®)and divalproex sodium (Depakote®). Swelling of the brain may be treatedwith steroids (e.g., dexamethasone (Decadron®).

D. Pharmaceutical Compositions

As mentioned above, methods of treatment, inhibition and/or reduction,and/or detection of the present invention include administration of achlorotoxin agent per se or in the form of a pharmaceutical composition.A pharmaceutical composition will generally comprise an effective amountof at least one chlorotoxin agent and at least one pharmaceuticallyacceptable carrier or excipient.

Pharmaceutical compositions may be formulated using conventional methodswell-known in the art. The optimal pharmaceutical formulation can bevaried depending upon the route of administration and desired dosage.Such formulations may influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of the administeredcompounds. Formulation may produce solid, liquid or semi-liquidpharmaceutical compositions.

Pharmaceutical compositions may be formulated in dosage unit form forease of administration and uniformity of dosage. The expression “unitdosage form”, as used herein, refers to a physically discrete unit ofchlorotoxin agent for the patient to be treated. Each unit contains apredetermined quantity of active material calculated to produce thedesired therapeutic effect. It will be understood, however, that thetotal dosage of the composition will be decided by the attendingphysician within the scope of sound medical judgment.

As mentioned above, in certain embodiments, the chlorotoxin agent isadministered intravenously through injection or infusion. Pharmaceuticalcompositions suitable for administration by injection or infusion may beformulated according to the known art using suitable dispersing orwetting agents, and suspending agents. The pharmaceutical compositionmay also be a sterile injectable solution, suspension or emulsion in anon-toxic diluent or solvent, for example, as a solution in2,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solution or suspension medium. For this purpose, any blandfixed oil can be used including synthetic mono- or di-glycerides. Fattyacids such as oleic acid may also be used in the preparation ofinjectable formulations.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from injection. This may be accomplished bydissolving or suspending the active ingredient in an oil vehicle.Injectable depot forms are made by forming micro-encapsulated matricesof the drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Depot injectable formulations can also be prepared by entrapping thedrug in liposomes or microemulsions that are compatible with bodytissues.

III. Methods of Detecting

A. Administration

In another aspect, the present invention provides methods for in vivodetection of tumor metastases. For example, it may be desirable to useinventive methods to detect tumor metastases in individuals who have orhave had at least one primary tumor. Such methods include administeringto a patient an effective amount of a labeled chlorotoxin agentdescribed herein, or a pharmaceutical composition thereof, such thatspecific binding of the labeled chlorotoxin agent to cells in primarytumor tissue and/or metastatic tumor tissue can occur.

Generally, the dosage of a labeled chlorotoxin agent will vary dependingon considerations such as age, sex, and weight of the patient, area(s)of the body to be examined, as well as the administration route. Factorssuch as contraindications, concomitant therapies, and other variablesare also to be taken into account to adjust the dosage of the labeledchlorotoxin agent to be administered. This can, however, be readilyachieved by a trained physician. In general, a suitable dose of alabeled chlorotoxin agent corresponds to the lowest amount of agent thatis sufficient to allow detection of neoplastic tumor tissue in thepatient.

For example, in embodiments where the chlorotoxin agent is labeled with¹³¹I and administered intravenously, dosing of the labeled chlorotoxinagent may comprise administration of one or more doses each comprisingabout 5 mCi to about 50 mCi, e.g., about 5 mCi to about 40 mCi, or about10 mCi to about 30 mCi ¹³¹I. For example, one or more doses of¹³¹I-radiolabeled chlorotoxin agent may be administered that eachcontain about 10 mCi, about 20 mCi, or about 30 mCi ¹³¹I. In suchembodiments, a diagnosis procedure may comprise administration of asingle dose of ¹³¹I-radiolabeled chlorotoxin agent or administration ofmultiple doses, e.g., 2 doses, 3 doses, or 4 doses. Two consecutivedoses may be administered at 1 day interval, 2 days interval, 3 daysinterval, 4 days interval, 5 days interval, 6 days interval, 7 daysinterval, or more than 7 days interval.

In embodiments where a ¹³¹I-radiolabeled chlorotoxin agent is used, thepatient may be administered supersaturated potassium iodide prior toadministration of the ¹³¹I-radiolabeled chlorotoxin (e.g., 1 day, 2days, or 3 days before treatment according to the present invention).Administration of supersaturated potassium iodide blocks uptake of ¹³¹Iby the thyroid gland, thus preventing conditions such as hypothyroidism.

Following administration of the labeled chlorotoxin agent and aftersufficient time has elapsed for specific binding to take place,detection of the bound labeled chlorotoxin agent is performed.

In some embodiments, a second effective amount of a labeled chlorotoxinagent is administered at a second time period and binding of the labeledchlorotoxin agent in the individual's body is measured. Measurement ofbinding after the second administration of labeled chlorotoxin agent mayallow assessment of any change in binding (e.g., in extent and/orlocation of binding), which may be indicative of progression, stability,and/or regression of one or more metastases. Subsequent (i.e., third,fourth, etc.,) administrations of effective amounts of a labeledchlorotoxin agent during subsequent time periods to obtain additionalmeasurements may also be performed. This may be desirable, for example,to assess the progression, stability, and/or regression of one or moremetastases over a longer period of time.

In some embodiments, the length of time between consecutiveadministrations of effective amounts of a labeled chlorotoxin agentvaries. In some embodiments, administrations of a labeled chlorotoxinagent are performed at approximately regular intervals. In someembodiments, timing of administration matches or parallels the timing ofa dosing regimen that the patient undergoes for treatment.

B. Metastasis Detection and Localization

As will be recognized by one skilled in the art, detection of binding ofa labeled chlorotoxin agent to a tissue of interest may be carried outby any of a wide variety of methods including, but not limited to,spectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Selection of a detection method willgenerally be based on the nature of the labeling moiety of the agent(i.e., fluorescent moiety, radionuclide, paramagnetic metal ion, and thelike). In certain embodiments, detection and localization of one or moremetastases within a patient are carried out using an imaging technique.

Different imaging techniques can be used depending on the nature of thelabeling moiety. For example, the binding may be detected using MagneticResonance Imaging (MRI) if the labeling moiety comprises a paramagneticmetal ion (e.g., Gd³⁺). Single Photon Emission Computed Tomography(SPECT) and/or Positron Emission Tomography (PET) can be used forbinding detection if the labeling moiety comprises a radioisotope (e.g.,¹³¹I, and the like). Other imaging techniques include gamma cameraimaging.

According to detection methods of the present invention, a tissue otherthan the tissue of the primary tumor is identified as a metastasis ifthe level of binding of the labeled chlorotoxin agent to the tissue ofinterest is elevated compared to the level of binding of the labeledchlorotoxin agent to a normal tissue. As already mentioned above, anormal tissue is herein defined as a non-neoplastic tissue. For example,when the method is performed in vivo, the level of binding of thelabeled chlorotoxin agent measured in a region of an organ of interest(e.g., the brain) may be compared to the level of binding of the labeledchlorotoxin agent measured in a normal region of the same organ.

In certain embodiments, the tissue of interest is identified as aneoplastic tissue if the level of binding measured is higher than thelevel of binding to a normal tissue. For example, the level of bindingmay be at least about 2 times higher, at least about 3 times higher, atleast about 4 times higher, at least about 5 times higher, at leastabout 10 times higher, at least about 25 times higher, at least about 50times higher, at least about 75 times higher, at least about 100 timeshigher, at least about 150 times higher, at least about 200 timeshigher, or more than 200 times higher than the level of binding to anormal tissue.

EXAMPLES

The following examples describe some of the modes of making andpracticing the present invention. However, it should be understood thatthese examples are for illustrative purposes only and are not meant tolimit the scope of the invention. Furthermore, unless the description inan Example is presented in the past tense, the text, like the rest ofthe specification, is not intended to suggest that experiments wereactually performed or data were actually obtained.

Example 1 Chlorotoxin Binds Melanoma Metastasized to the Brain and Lung

The experiments described in this Example demonstrate that chlorotoxinbinds to melanoma that has metastasized to the brain and/or the lung inbiopsy sections.

Materials and Methods

Frozen or paraffin sections of human biopsy tissues were histochemicallystained with a chemically synthesized form of chlorotoxin containing adetectable biotin group chemically attached to the N terminus (TM-601).Samples of human tissue were from both sexes and different ages andraces. Most samples were obtained through the Cooperative Human TissueNetwork, Tissue Procurement at UAB (the University of Alabama atBirmingham), UAB hospitals, and the Human Brain Tissue Bank in London,Canada. Snap frozen tissue and fresh tissue embedded in a freezing gelwere sliced at 8 microns and picked up onto positively charged glassslides. The sections were then fixed in 4% paraformaldehyde or Milloniqssolution (composed of 4% formaldehyde, 0.4% NaOH, and 7% methanol in abuffered sodium phosphate solution) according to the staining protocol.Paraffin blocks were sectioned and prepared according to standardprocedures.

Biopsy sections were blocked for 1 hour in 10% normal goat serum in PBSand treated with a dilution of biotinylated chlorotoxin overnight at 4°C. After thorough rinsings, the stainings were developed by theavidin-biotin complex (ABC) system (Vectastain Elite ABC Kit from VectorLaboratories, Burlignton, Calif.) and visualized by the colorimetricreaction of DAB (3,3′-diaminobenzidine, Vector Laboratories) with theABC complex.

The biopsy sections were counterstained with methyl green, a nucleardye, to visualize unstained cells. Non-specific background label canvary from experiment to experiment due to changes in the effectiveconcentration of the label, condition of the tissue, or the duration ofthe reaction. Therefore, a control section was identically stained withmethyl green but without the biotinylated chlorotoxin. Positive cellstaining was defined by chlorotoxin-labeling above background whencompared to an adjacent control section. Cells containing high amountsof endogenous peroxidase exhibit dark background staining in thecontrols due to the reaction of DAB with the peroxidases.

A third adjacent section was stained with both hematoxylin (which stainscell nuclei) and eosin (which stains cytoplasm). Therefore, for eachtissue analyzed, three adjacent sections were stained.

Results

FIG. 1 depicts photomicrographs showing that biotinylated chlorotoxinstains melanoma metastasized to the brain. Eleven out of 11 melanomabrain metastases were positive for TM-601, and 5 out of 5 primarymelanoma tumors were positive. In addition, chlorotoxin also binds tomelanoma metastasized to the lung (FIG. 2). On the other hand, normalskin is unreactive to TM-601 (6/6 negative) (FIG. 3), although there issome background staining in melanocytes even in controls.

Example 2 Phase I Imaging and Safety Study of Intravenous ¹³¹I-TM-601 inPatients with Recurrent or Refractory Somatic and/or Cerebral MetastaticSolid Tumors

The present Example describes preliminary results from a Phase I trialconducted at 5 clinical sites. In this clinical trial, TM-601 wasadministered intravenously to 48 patients. This multi-center,open-label, non-randomized, sequential “within subject” escalation studyincluded patients with histologically confirmed primary solid tumormalignancy, either recurrent or refractory, who had demonstratedunequivocal evidence of detectable metastatic involvement that was notamenable to standard therapy.

The objectives of this Phase I study were: a) to evaluate whetherintravenous ¹³¹I-TM-601 provides tumor-specific localization in patientswith recurrent or refractory metastatic (including brain metastases)solid tumors; b) to determine the distribution and dosimetry ofintravenously administered ¹³¹I-TM-601; and c) to determine the safetyand tolerability of intravenously administered ¹³¹I-TM-601.

Patients and Treatment Protocol

Forty-eight subjects were enrolled in this study. Protocols describedbelow were used with these subjects. Subjects underwent 1 to 2escalating intravenous doses of ¹³¹I-TM-601 followed by a series ofwhole body scans to determine whether the ¹³¹I-TM-601 has localized totarget tumor cells, and one intravenous therapeutic dose of ¹³¹I-TM-601once tumor-specific uptake of ¹³¹I-TM-601 was demonstrated. The graphicin FIG. 4 illustrates the dosing scheme.

Study patients received up to three doses of ¹³¹I-TM-601 (ranging from10 mCi/0.2 mg to 30 mCi/0.6 mg) by intravenous (IV) infusion. Onlypatients demonstrating tumor specific uptake of ¹³¹I-TM-601 by imagingperformed 24 hours after administration of the 10 or 20 mCi dose wereadministered the 30 mCi dose of ¹³¹I-TM-601.

Preparation of ¹³¹I-TM-601

The final TM-601 drug product is a sterile, lyophilized white tooff-white powder vialed in stoppered glass vials. The imaging andtherapeutic doses used in this trial were doses of radio-labeled TM-601.

TM-601 final drug product was reconstituted in 0.56 mL of radio-labelingbuffer to yield a 1 mg/mL solution radio-labeled with ¹³¹I, anddelivered to the clinical site. The syringe contained approximately 4 mLof solution for infusion and was approximately labeled as to content andamount of radioactivity. Once received at the site, the radiation safetyofficer or other appropriate site personnel confirmed that the radiationcount of the ¹³¹I-TM-601 was within prescribed specifications. Thesyringe containing the final radio-labeled drug product was shielded andthen transferred to the appropriate hospital area for administration tothe patient. The ¹³¹I-TM-601 solution was stored protected from light at2-8° C. and shielded until use. After radio-labeling with ¹³¹I, theproduct was recommended to be used within 24 hours.

Administration of ¹³¹I-TM-601 and Imaging Study

All patients receiving the radio-labeled test dose, ¹³¹I-TM-601,received supersaturated potassium iodide (SSKI) at a dose of 300 mg/dayorally, beginning on the day of and just prior to radio-labeled¹³¹I-TM-601 infusion and for a minimum of three days to block uptake of¹³¹I to the thyroid and other organs. SSKI was dispensed to the patientprior to study drug administration with instructions provided to thepatient on the proper use of the drug while not in the clinic/hospital.

The syringe containing ¹³¹I-TM-601 was inserted “piggy-back” fashioninto an infusion port within six-inches of the intravenousneedle/catheter. While running 0.9% sodium chloride at 100 mL/hour, theproduct was administered by “slow IV push” over approximately 5-10minutes. ¹³¹I-TM-601 infusion was terminated if any of the followingwere observed: (1) a fall in systolic blood pressure >25 mmHg, (2) asignificant respiratory distress documented by the investigator, (3)temperature >102° F., (4) seizures, (5) changes in level ofconsciousness or onset of new neurological deficit, or other reasons,such as clinician's judgment or patient's request.

Imaging by gamma camera and in some cases SPECT was performed 24 hourspost ¹³¹I-TM-601 administration to determine localization andeligibility for receiving the 30 mCi dose of ¹³¹I-TM-601.

Safety Results

As of May 2008, a total of 22 serious adverse events were reported for17 patients. All SAEs were judged by the Investigator to be “unlikely”or “unrelated” to the study drug and four of these events occurred intwo patients following consent who did not go on to receive ¹³¹I-TM-601study drug. A fifth event occurred in a patient prior to receipt ofstudy drug who later was successfully dosed.

Efficacy Results

Tumor specific uptake was observed in a variety of tumor types followingintravenous administration, including seven out of eight patients withmalignant glioma; two out of two patients with metastatic prostatecancer including one with diffuse bone metastases; three out of fivepatients with metastatic non-small cell lung cancer; seven out of eightpatients with metastatic melanoma; six out of eight patients withmetastatic colon cancer; two of three patients with metastaticpancreatic cancer; one out of four patients with metastatic breastcancer; one patient with metastatic transitional cell carcinoma; onepatient with metastatic paraganglioma, and one patient with pleomorphicxanthoastrocytoma (as summarized in FIG. 5; see also FIGS. 6-9).

All patients received a test dose of 10 mCi (0.2 mg peptide) ¹³¹I-TM-601intravenously. Five sequential, whole body gamma camera images wereacquired at immediate ≦60 minutes), 3 hours, 24 hours, 48-72 hours, and168 hours post ¹³¹I-TM-601 injection for tumor localization anddosimetry analysis. Patients showing tumor localization by gamma cameraor SPECT imaging received a second therapeutic dose of 30 mCi (0.6 mgpeptide) ¹³¹I-TM-601 one week later. Patients not showing uptake werere-treated a week later with 20 mCi (0.4 mg peptide) ¹³¹I-TM-601 todetermine possible localization at a higher dose.

Tumor response (as defined by a decrease in the volume ofgadolinium-enhancing disease) has been seen in magnetic resonanceimaging (MRI) of two of the eight glioma patients at the day 28evaluation, which demonstrated a measurable reduction in tumor volumefrom baseline. (see FIGS. 10 and 11).

These results demonstrate the therapeutic effect of chlorotoxin agentssuch as ¹³¹I-TM-601 delivered systemically in vivo. These results alsodemonstrate that ¹³¹I-TM-601 administered intravenously will cross theblood brain barrier and can result in MRI imaging improvement inpatients with inoperable gliomas. Furthermore, in patients withmetastatic cancers, ¹³¹I-TM-601 delivered intravenously is able totarget distant metastases (see, e.g., FIG. 12).

Example 3 Tumor-Specific Targeting of Intravenous ¹³¹I-Chlorotoxin(¹³¹I-TM-601) in Patients with Metastatic Melanoma

In previous clinical trials, patients with recurrent glioblastomamultiforme were administered ¹³¹I-chlorotoxin locally into a tumorresection cavity. In the present Example, distribution of intravenously(IV) administered ¹³¹I-chlorotoxin was examined to determine if theintravenous route of administration would be feasible and would resultin intratumoral uptake in patients with metastatic melanoma, includingmetastases to the CNS. The experiments described in this Exampledemonstrate that ¹³¹I-chlorotoxin delivered intravenously localizes totumor sites throughout the body as well as to metastases in the brain.Thus, ¹³¹I-chlorotoxin administered intravenously crosses the bloodbrain barrier and may be used to target distant metastases.

Materials and Methods

Seven patients with metastatic melanoma were enrolled in the prospectiveclinical trial of systemically administered ¹³¹I-chlorotoxin discussedin Example 2. The present Example discusses in greater detail theresults with these seven patients. All patients received a test dose of10 mCi (0.2 mg peptide) ¹³¹I-chlorotoxin intravenously. Five sequentialwhole body gamma camera images were acquired immediately (≦60 minutes)and 3 hours, 24 hours, 48-72 hours, and 168 hours post ¹³¹I-chlorotoxininjection for tumor localization and dosimetry analysis. Patientsshowing tumor localization by gamma camera or SPECT imaging received asecond therapeutic dose of 30 mCi (0.6 mg peptide) ¹³¹I-chlorotoxin oneweek later. Patients not showing uptake were re-treated a week laterwith 20 mCi (0.4 mg peptide) ¹³¹I-chlorotoxin to determine possiblelocalization at a higher dose.

Results

Six of the seven enrolled patients with melanoma demonstratedtumor-specific localization on follow-up gamma camera or SPECT imagingafter intravenous administration of ¹³¹I-chlorotoxin. Tumor localizationwas observed in the central nervous system and at extracranial sites.(See FIG. 13 for an example.) The remaining patient was withdrawn fromstudy following the first test dose (10 mCi/0.20 mg peptide) and was notconsidered evaluable. Dose limiting toxicity was not observed. Fulldosimetric analysis was available on three patients treated at theUniversity of Alabama at Birmingham. The mean radiation dose wasapproximately 0.24 cGy/mCi (ranging from approximately 0.21 toapproximately 0.27 cGy/mCi) to total body and approximately 2.56 cGy/mCi(ranging from approximately 1.36 to approximately 4.43 cGy/mCi) to tumorwith a calculated therapeutic ratio of approximately 10 (tumor dose/bodydose).

The results demonstrate that ¹³¹I-chlorotoxin administered intravenouslycrosses the blood brain barrier and produces a high rate of tumorspecific targeting. Thus, ¹³¹I-chlorotoxin may be used to target distantmetastases including those in the brain. Future clinical trials willevaluate the safety and efficacy of higher doses of intravenouslyadministered ¹³¹I-chlorotoxin in a variety of tumor types.

Example 4 Inhibition and Regression of Choroidal Neovascularization byTM-601

The formation of new blood vessels (angiogenesis) and maintenance ofsuch blood vessels is thought to be an important element of metastasis.In the present Example, the ability of chlorotoxin to inhibitangiogenesis and/or cause regression of existing newly formed bloodvessels was evaluated using a choroidal neovascularization assay. WhenTM-601 was administered beginning around the time of induction of bloodvessel formation, TM-601 caused a significant decrease in new bloodvessel formation. In an experimental paradigm in which TM-601 wasadministered several days after blood vessel formation was induced,TM-601 caused significant regression of choroidal neovascularization.

Materials and Methods

Choroidal neovascularization (CNV) was induced in mice byphotocoagulation with a 530 nm laser. Three burns were delivered to eachretina in the 9, 12, and 3 o'clock positions of the posterior pole ofthe retina. Rupture of Bruch's membrane was judged to be successful whena bubble was produced at the time of laser induction. Only burns forwhich bubbles were observed were included in this study.

In the first experiment, intravitreal injection of 1 μL, of a 50 mg/mLsolution of TM-601 dissolved in saline was injected in one eye (n=17animals, 49 quantifiable burns) and 1 μL of saline was injected in thefellow eye following laser photocoagulation (n=17 animals, 44quantifiable burns). Seven days later, the injections were repeated. Onday 14 of the study, mice were perfused with fluorescein-labeled dextran(2×10⁶ average molecular weight, Sigma) and choroidal flat mounts wereprepared and examined by fluorescence microscopy.

In the second experiment, laser photocoagulation was performed on day 1.One group of 10 mice (30 quantifiable burns) were perfused withfluorescein-labeled dextran on day 7 for CNV baseline measurement priorto treatment initiation. The remainder of the mice received anintraocular injection of 1 μL of a 50 mg/mL solution of TM-601 in oneeye (n=12 animals, 34 quantifiable burns) and saline in the fellow eye(n=13 animals, 32 quantifiable burns). On day 14, all remaining micewere perfused with fluorescein-labeled dextran and choroidal flat mountswere prepared and examined by fluorescence microscopy.

Sizes of choroidal neovascularization lesions were measured in choroidalflat mounts. After perfusion with fluorescein-labeled dextran, eyes wereremoved and fixed for 1 hour in 10% buffered formalin. The cornea andlenses were removed and the entire retina was dissected from the eyecup. Radial cuts of the choroids were made from the edge to the equatorand the eyecup was flat mounted. Flat mounts were examined byfluorescence microscopy and images were digitized. Image-Pro Plussoftware (Media Cybernetics) was used to measure the total area ofchoroidal neovascularization associated with each burn.

Results

Rupture of Bruch's membrane with laser photocoagulation in mice causeschoroidal neovascularization (CNV), which mimics many aspects of CNVthat occurs in patients with neovascular age-related maculardegeneration. To determine whether TM-601 impacts the formation of newblood vessels in this model, intravitreal injections of 50 μg TM-601were performed on the day of laser photocoagulation (day 1) and on day7. Control eyes were injected with saline at the same time points.Fourteen days after rupture of Bruch's membrane, choroidal flat mountsfrom each eye were analyzed. TM-601 treatment was found to significantlydecrease the formation of new blood vessels with intraocular TM-601doses of 50 μg (FIGS. 13 and 15).

To assess the effect of TM-601 on pre-existing neovasculature in thismodel, treatment with intraocular injection with 50 μg TM-601 wasdelayed until 7 days after disruption of Bruch's membrane. At this timepoint, large sites of neovascularization were already present (seebaseline in FIG. 14). A single saline injection on day 7 had no effecton new blood vessel formation measured on day 14 (control in FIG. 14),whereas a single injection of TM-601 significantly caused regression ofCNV (FIGS. 14 and 15).

Discussion/Conclusion

The present Example demonstrates that locally administered TM-601 cansignificantly suppress CNV and cause regression of CNV. The CNV mousemodel mimics the disease state for the wet form of macular degeneration.The intravitreal route of administration used in this study may beclinically relevant, since it is the route used for administeringLucentis®, a clinically approved therapy for macular degeneration.

Example 5 TM-601 Inhibits Angiogenesis in a Mouse CNV Model Via VariousRoutes of Delivery

The experiments described in the present Example were conducted toevaluate anti-angiogenic ability of TM-601 delivered by various routesof administration. A choroidal neovascularization assay was used tomeasure new blood vessel growth around sites of laser-induced rupture ofBruch's membrane and to determine whether local or systemicadministration of TM-601 cause decreased angiogenesis. Three new routesof administration were tested: periocular (also referred to assubconjunctival), intravenous, and topical (eye drops).

Materials and Methods

Choroidal neovascularization (CNV) in mice was induced by 530 nm laserphotocoagulation. Three burns were delivered to each retina in the 9, 12and 3 o'clock positions of the posterior pole of the retina. Successfulrupture of Bruch's membrane was evident when production of a bubbleoccurred at the time of the laser induction. Only burns in which abubble was observed were included in the study.

Dosing

Periocular injections of 5 μL TM-601 solution were performed with TM-601concentrations of 2, 10, 50 and 200 mg/mL dissolved in saline. Fivemicroliters of saline was injected in the fellow eye following laserphotocoagulation. Seven days later, injections were repeated. On day 14of the study, mice were perfused with fluorescein-labeled dextran (2×10⁶average molecular weight, Sigma) and choroidal flat mounts were preparedand examined by fluorescence microscopy.

Intravenous dosing was performed by tail vein injections at a dose of 20mg/kg TM-601 three times per week.

Topical application of TM-601 was achieved by application of eye dropsthree times a day, each eye drop containing a volume of 10 μL TM-601.TM-601 was dissolved in over-the-counter Artificial Tears (Rite Aide)which contains as active ingredients 70% dextran and 0.3% hypromellose.Inactive ingredients include 0.1% benzalkonium, edetate disodium,potassium chloride, purified water and sodium chloride. The finalconcentrations of TM-601 were 1, 5 and 25 mg/mL to give a single dose of10, 50, and 250 μg respectively per 10 μL drop.

Histology and Imaging

Sizes of choroidal neovascularization lesions were measured in choroidalflat mounts. After perfusion with fluorescein-labeled dextran, the eyeswere removed and fixed for 1 hr in 10% buffered formalin. Cornea andlenses were removed and the entire retina was dissected from the eyecup. Radial cuts of the choroids were made from the edge to the equatorand the eyecup was flat mounted. Flat mounts were examined byflorescence microscopy and images were digitized. Image-Pro Plussoftware (Media Cybernetics) was used to measure the total area ofchoroidal neovascularization associated with each burn.

Results

Rupture of Bruch's membrane with laser photocoagulation in mice causeschoroidal neovascularization (CNV), which mimics many aspects of CNVthat occurs in patients with neovascular age-related maculardegeneration. Using this model, the inventors had previously shown thatintraocular injections of TM-601 significantly decreasesneovascularization and also causes regression of new blood vessels. (SeeExample 4).

In the current study, other routes of administration (periocular,intravenous and topical) were examined. In the first periocular study,periocular injections of 250 μg TM-601 were performed on the day oflaser photocoagulation (day 1) and on day 7. Control eyes were injectedwith saline at the same time points. Fourteen days after rupture ofBruch's membrane, choroidal flat mounts from each eye were analyzed.TM-601 treatment was found to significantly decrease the formation ofnew blood vessels with periocular TM601 doses of 250 μg (FIG. 16).

To determine the dose response for periocular injections, TM-601 wasinjected at a dose of 10 μg, 50 μg, 250 μg or 1000 μg. The 10 μg dosedid not significantly decrease choroidal neovascularization, but dosesof 50 μg or greater reduced CNV to a similar extent (FIG. 17).Interestingly, fellow eyes in animals that received periocularinjections also exhibited reductions in CNV (green curve in FIG. 17).

To determine whether systemically injected TM-601 would also penetrateinto the choroid and inhibit angiogenesis, TM-601 was injected by tailvein three times per week over the course of the two week study at adose of 20 mg/kg. With intravenous injections, TM-601 was found tosignificantly reduce CNV (FIG. 18).

Topical eye drop application, the least invasive of the three routes ofadministration examined in this Example, was also tested. TM-601 wasresuspended in an over-the-counter eye drop lubricant and was appliedthree times per day via a 10 μL eye drop. At the highest dose delivered(0.25 mg/drop, three times per day), a decline in CNV was observed, butit was not statistically different from the saline control (FIG. 19).

Discussion/Conclusion

The CNV mouse model mimics the disease state for the wet form of maculardegeneration. As shown in Example 4, an intravitreal route ofadministration can significantly decrease choroidal neovascularization.This is the same route used for administering Lucentis®, a clinicallyapproved therapy for macular degeneration. However, the inventorsrecognized that a less invasive mode of delivery may be advantageous andtested periocular, intravenous and topical delivery of TM-601. In allcases, TM-601 was shown to decrease CNV, although the decrease seen withtopical drops was not observed to reach statistical significance.Periocular injections of TM-601 may be advantageous over drugs thatrequire intraocular delivery (e.g. Lucentis®).

The “fellow-eye effect” observed in FIG. 17 may be interesting. TM-601injected periocularly caused significant reductions in CNV in theadjacent eye (which did not receive injections). Without wishing to bebound by any particular theory, a possible explanation for thisphenomenon is that the injected material entered systemic circulation,thus resulting in drug exposure to the fellow eye. A similar fellow-eyeeffect was not observed with topical eye drops.

These results indicate that TM-601 delivered by different routes ofadministration showed anti-angiogenic effects, thus providing furthersupport for chlorotoxin's utility as a therapeutic agent for metastaticcancers.

Example 6 Localization of TM-601 to New Blood Vessels and TM-601-InducedApoptosis of Neovascular Endothelial Cells

Experiments described in Examples 4 and 5 demonstrated that chlorotoxincan inhibit angiogenesis and cause regression of new blood vessels in amouse model of choroidal neovascularization (CNV). Experiments describedin the present Example were directed to understanding the mechanism ofchlorotoxin's anti-angiogenic effects as demonstrated in Examples 4 and5. Results in the present Example demonstrated that TM-601 localizes tonew blood vessels and induces apopotosis of neovasucular endothelialcells in a mouse CNV model. Furthermore, results in the present Exampledemonstrated that TM-601 colocalizes with Annexin A2, which theinventors have observed in previous studies to bind to TM-601, in areasof neovascularization in both a CNV model and a retinopathy model.

Although TM-601 was shown to inhibit choroidal neovascularization andregression of newly formed vessels in the eye, it was not previouslyknown whether TM-601 directly bound to a particular cell type in theeye, or was pharmacologically active in a non-specific manner.Generalized binding to the vasculature was not observed in whole bodyplanar images after intravenous injection of ¹³¹I-TM-601. It wastherefore hypothesized, without wishing to be bound by any particulartheory, that TM-601 selectively binds only to a subset of activated orproliferating cells at a site of new blood vessel formation.

New blood vessel formation was induced in a mouse model of CNV, and thelocation of TM-601 after intravitreal or subconjunctival injection insuch a model was determined. In addition, a TUNEL assay was used todetermine whether regression of new blood vessels was due to apoptosisof endothelial cells at the site of choroidal neovascularization.

To further understand the mechanism of TM-601's anti-angiogenic effects,colocalization of TM-601 with chlorotoxin's likely cellular receptor,Annexin A2, in areas of neovascularization was examined byimmunohistochemistry. Annexin A2 has been observed by the inventors tobind to TM-601. Annexin A2 is expressed on surfaces of endothelial cellsand is overexpressed in certain tumor types. Annexin A2 has beencharacterized as a docking station that regulates the conversion of pro-and/or anti-angiogenic proteins such as plasminogen and plasmin.

Materials and Methods

Choroidal neovascularization (CNV) in mice was induced by 530 nm laserphotocoagulation. Three burns were delivered to each retina in the 9, 12and 3 o'clock positions of the posterior pole of the retina. Successfulrupture of Bruch's membrane was evident when production of a bubbleoccurred at the time of the laser induction. Only burns in which abubble was observed were included in the study.

Dosing

Intraocular injections of 1 μL TM-601 (50 μg/μL dissolved in saline)were performed on day 7 following CNV lesion. Subconjunctival injectionsof 5 μL were performed on days 7 and 8 using a TM-601 concentration of10 μg/μL dissolved in saline. No injections were given to the felloweye. Animals were sacrificed on day 9, the eyes were removed, andsections were cut through CNV lesions. Frozen sections were stained asdescribed below.

Immunohistochemistry

For TM-601 localization studies, rabbit anti-TM-601 (red in FIGS. 20A,B, and C and FIGS. 21A, B, and C) primary antibody was used.Fluorescence detection was performed using a fluorescent-taggedanti-rabbit IgG secondary antibody. Sections were also stained withfluorescently tagged GSA lectin (green in FIGS. 20D, E, and F and FIGS.21D, E, and F) to identify endothelial cells. For detection of apoptoticcells, sections were stained for Terminal deoxynucleotidyl TransferasedUTP Nick End Labeling (TUNEL). This assay is used to detect cell nucleithat exhibit DNA fragmentation, a hallmark of apoptotic cell death.Sections were also stained with a nuclear stain. For colocalizationstudies with Annexin A2, sections were also stained with anti-AnnexinA2.

Endothelial Cell Proliferation Assay

Endothelial cell proliferation assay was performed using the CellTiter96® Aqueous One Solution Cell Proliferation Kit from Promega (cataloguenumber #G3582). Briefly, either 4,000 cells (72 hr assay) or 1,000 cells(120 hr assay) were plated overnight in each well of a 96 well tray. Thenext day, TM-601 or diluent was added to the wells. The cells were thencultured for either 72 or 120 hrs at 37° C. Cell number was determinedby staining with MTS tetrazolium according to kit protocol. The quantityof formazan product as measured by the absorbance at 490 nm is directlyproportional to the number of living cells in culture.

Results

To investigate the mechanism of TM-601's antiangiogenic effects in amouse model of CNV, localization of TM-601 was studied by injectingTM-601 intraocularly into mouse eyes on day 7 or periocularly on days 7and 8 after laser-induced photocoagulation. On day 9 of the study, eyeswere immunostained for TM-601. With both routes of administration,TM-601 was found to specifically localize to endothelial cells in thechoroid (FIGS. 20 and 21). No detectable TM-601 was observed to beassociated with pre-existing vessels below the retinal layer, indicatingthat TM-601 bound selectively to newly formed vessels in the choroid.

As described in Example 4, one week after new blood vessel formation asa result of laser photocoagulation, a single intraocular injection ofTM-601 caused significant regression of CNV. However, the mechanism ofthis effect was unknown. To test whether TM-601 could cause apoptosis ofcells that contribute to the newly forming vasculature in this model,sections cut through CNV lesions were stained for TUNEL. Apoptotic cells(as identified by positive TUNEL staining) co-localized with anendothelial stain, indicating that endothelial cells in the region ofchoroidal neovascularization were undergoing apoptosis following eitherintraocular or periocular TM-601 treatment (FIGS. 22 and 23). Noapoptosis was detected in eyes injected with saline. Apoptosis ofendothelial cells in treated eyes in vivo was unexpected because invitro treatment of cultured endothelial cells with TM-601 is notcytotoxic over a wide range of TM-601 concentrations (FIG. 24).

To further understand the mechanism of TM-601's anti-angiogenic effects,colocalization studies were conducted with TM-601 and Annexin A2,chlorotoxin's likely cellular receptor. Neovascularization was inducedin the eye in two different models, choroidal neovascularization inducedby laser disruption of Bruch's membrane and retinopathicneovascularization induced by oxygen-induced ischemia. TM-601 wasinjected intraocularly as described herein for similar experients. Inboth models of neovascularization, TM-601 colocalized with Annexin A2(FIG. 25).

Discussion/Conclusion

Results presented in this Example support several important findingsrelated to chlorotoxin's anti-angiogenic effects. First, TM-601 injectedintraocularly or periocularly selectively binds to endothelial cells innewly formed vasculature in the choroid layer after laser-inducedrupture of Bruch's membrane. TM-601 did not bind to pre-existing maturevessels. These results are consistent with the observation ¹³¹I-TM-601does not indiscriminately bind to all vessels in the body followingintravenous injection. Instead, radiolabel is detected predominantly inthe region of the tumor due to tumor-specific and/orneovascular-specific binding. Second, binding of TM-601 to endothelialcells results in selective apoptosis of new vessels forming around thesite of laser injury, but not in pre-existing vessels. Third, TM-601colocalizes with Annexin A2, which is involved in regulating theconversion of pro- and/or anti-angiogeneic proteins such as plasminogenand plasmin, raising the possibility that chlorotoxin exerts at leastsome of its anti-angiogenic effects through Annexin A2.

Example 7 Effect of TM-601 on Tumor Cell Migration In Vitro

Experiments described in this Example were directed to elucidating therole of chlorotoxin on cell migration, which plays a major role inmetastasis. TM-601 showed a dose-dependent inhibitory effect on cellmigration, as assessed by a Transwell invasion assay.

Materials and Methods

Migration of Human Umbilical Vein Endothelial Cells (HUVEC) across aTranswell (Corning, 8 μm) was performed on 5×10⁴ serum-starved cells intriplicate. Chemoattractants in the bottom well included VEGF or bFGF(50 ng/ml) in media containing 0.4% FBS. Ten micromolar TM-601 wasincubated with the cells for 30 min at room temperature prior to loadingcells into the Transwell. After 22 hrs at 37° C., non-migrated cells onthe upper surface were removed using a Q-tip. Cells that migratedthrough the membrane were fixed in methanol and visualized with Giemsastain. Quantitative counts of invaded cells were performed bysuperimposing the Transwell over a hemocytometer and counting the numberof cells in each of five regions.

Results

As shown in FIG. 26A, TM-601 inhibited cell migration in adose-dependent manner. Approximately fifty-percent Fewer cells migratedin the presence of TM-601 whether they were stimulated by VEGF or bFGF(FIG. 26B).

Example 8 Effect of TM-601 on MMP2 Activity

As discussed previously, cell migration plays a major role inmetastasis. Degradation of the extracellular matrix facilitates cellmovement and is a key step in cell migration. In the present example,the effect of TM-601 on matrix metalloproteinase 2 (MMP2), an enzymethat degrades the extracellular matrix, was studied in two differentcell types: HUVECs and U87, a glioma cell line. Results from theseexperiments demonstrated that TM-601 inhibits MMP2 activity.

MMP-2 activity was measured in media taken from cultured HUVEC cellswithout treatment, treatment with bFGF, or treatment with bFGF togetherwith 10 μM TM-601. (HUVEC cells were cultured as described in Example7.) As depicted in FIG. 26C, treatment with TM-601 nearly obliteratedthe increase of MMP2 activity induced by bFGF.

MMP-2 activity was also measured in media taken from cultured U87 cellstreated with 10 μM TM-601 or with no TM-601. As depicted in FIG. 27,TM-601 decreases MMP-2 activity secreted from U87 human glioma cells.

These results suggest that TM-601 has an inhibitory effect on MMP-2activity.

Discussion for Examples 6-8

Taken together, data presented in Examples 6-8 provide a betterunderstanding of the anti-angiogenic effects of TM-601. In cell culture,TM-601 has been shown to bind to proliferating endothelial cells andblock cell migration (see Example 7), a critical step in the formationof new blood vessels. Without wishing to be bound by any particulartheory, it appears that some form of cellular activation is required invivo for binding of TM-601 to endothelial cells because only newlyformed vessels bind TM-601, whereas quiescent cells in the maturevasculature do not. In addition, proliferating HUVEC cells in culture donot mimic activated endothelial cells of the neovasculature becauseTM-601 does not decrease the proliferation of HUVEC cells (as would beexpected if apoptosis were occurring in vitro). Therefore, the datasuggest that one effect of TM-601 on newly forming vasculature is toinhibit formation of new vessels by blocking endothelial cell migration.TM-601 has been observed to exert a second effect in CNV models: newlyformed blood vessels that bind TM-601 undergo apoptosis.

In summary, in vitro evidence suggests that TM-601 blocks endothelialcell migration, thus preventing a key early step in angiogenesis. Inaddition, TM-601 has been shown to cause regression of newly formedvessels via apoptosis in a CNV model.

Results described in these Examples shed light on the mechanism ofchlorotoxin's anti-angiogenic properties, which are particularlyattractive qualities as regards chlorotoxin's utility as a therapeuticagent against metastatic tumors.

Example 9 Effect of Intravenously Delivered Unlabeled TM-601 on Tumorsin Human Patients

Human clinical data presented in this Example examine the ability ofintravenously delivered unlabeled TM-601 to treat cancer. As previouslydiscussed, in vitro data demonstrated that TM-601 binds to vascularendothelial cells and blocks endothelial cell migration. The inventorshad also obtained in vivo data demonstrating that intravenous infusionof TM-601 decreases neovascularization. These observations promptedinitiation of a phase I trial to evaluate the use of intravenouslydelivered unlabeled TM-601 in an invasive cancer with knownhypervascularity, malignant glioma. Primary objectives of this studyare: a) to determine the safety and tolerability of TM-601 in adultpatients with recurrent malignant glioma; b) to determine the targetrecommended phase II dose and biologically active dose of TM-601 whenadministered intravenously based on magnetic resonance (MR) perfusionimaging changes; and c) to determine the pharmacokinetics of TM-601 ateach dose level.

Patients and Treatment Protocol

Patients with recurrent malignant glioma eligible for this study areadministered 10 mCi/0.2 mg of ¹³¹I-TM-601 by intravenous (IV) infusionto demonstrate tumor specific localization (imaging dose). Only patientsdemonstrating tumor specific uptake of ¹³¹I-TM-601 on a brain SPECT scanremain on study to receive treatment with non-labeled TM-601. One weekafter the imaging dose, study patients receive non-labeled TM-601 byintravenous infusion at one of six dose levels (0.04 mg/kg, 0.08 mg/kg,0.16 mg/kg, 0.3 mg/kg, 0.6 mg/kg, and 1.2 mg/kg) once per week for 3weeks in a 4 week cycle. Subsequent cycles of non-labeled TM-601 areadministered as long as there is no evidence of disease progression andthe patient experiences no dose-limiting toxicities. Patients areevaluated at week 4 of each cycle with conventional and dynamicsusceptibility contrast MRI to assess perfusion.

As of January 2009, six patients with recurrent malignant glioma hadbeen enrolled in the study and had imaging data available for analysis.With respect to safety, there had been one event of intratumoralhemorrhage which was considered to be possibly related to therapy. Threeother serious adverse events considered to be unrelated to therapyincluded disease progression on therapy, a hip fracture, and renalcalculi in a patient with a history of renal calculi. Two of sixpatients in this first dosing cohort demonstrated a greater than 25%reduction in relative cerebral blood flow (rCBF) and/or relativecerebral blood volume (rCBV) compared to pre-treatment baseline. Bothpatients with improvement in perfusion MRI parameters had extendedresponse to intravenous TM-601 marked by multiple cycles of therapywithout evidence of tumor progression.

These results indicate that unlabeled chlorotoxin shows promise as atherapeutic against an invasive cancer with strong metastatic potential,which in the present Example is malignant glioma.

Example 10 Bioavailability and Anti-Angiogenic Effects of PEGylatedChlorotoxin

In the present Example, PEGylated chlorotoxin was studied to determineif the half-life of chlorotoxin in vivo could be increased.Anti-angiogenic effects of PEGylated chlorotoxin was also examined.

Materials and Methods

PEGylation

TM-601 was PEGylated at the N-terminus of the peptide via reductiveanimation using a polydispersed, linear, 40 kDa PEG-propionaldehyde(DowPharma).

Half-Life Measurements of TM-601

Non-tumor-bearing C57BL/6 mice were injected with TM-601 (at a dose ofapproximately 2 mg/kg) intravenously by a single tail vein injection.Blood samples were obtained at various timepoints, and levels of TM-601were determined by ELISA using an anti-TM-601 antibody.

Mouse Matrigel Plug

Matrigel Matrix High Concentration (from BD Biosciences) was mixed with100 ng/ml VEGF, 100 ng/ml bFGF, and 3 ng/ml heparin at 4° C. Eight-weekold female C57BL/6 mice were randomly assigned to each groups with 6mice in each group. Each mouse received two 500 μL Matrigel plugsinjected bilaterally in subcutaneous tissue. To form a round shapedplug, a wide subcutaneous pocket was formed by swaying the needlepointright and left after a routine subcutaneous insertion. The injection wasperformed rapidly with a 21-25 G needle to ensure the entire contentswere delivered into the plug. Matrigel plugs were implanted on Day 0 ofthe study and treatment began on Day 1. Animals were dosed withintravenous injections with either vehicle (saline), TM-601, orPEGylated TM-601. Three dosing regimens were used: once a week for twoweeks (once on D1 and once on D8; “Q7D×2”), twice a week for two weeks(on D1, D4, D8, and D11; “Q3D×2/2”), and five times a week for two weeks(on D1, D2, D3, D4, D5, D8, D9, D10, D11, and D12; “Q1D×5/2”). Plugswere collected after 14 days. Mice were euthanized and the skin over theplugs was pulled back. Plugs were dissected out, fixed, and embedded inparaffin for histological analysis. Three sections of 5 μm thicknessfrom each evaluable plug were immunostained with a CD31 antibody andcounterstained with hematoxylin & eosin. Blood vessel counts in a crosssectional area of each matrigel plug was analyzed under a microscope.

Results/Discussion

As shown in FIG. 28, PEGylated TM-601 exhibited an increased half-lifein vivo as compared to unmodified TM-601. PEGylation increased the halflife of TM-601 approximate 32-fold, that is, approximately 25 minutes(TM-601) to approximately 16 hrs (TM-601-PEG).

Increased half life translated into the ability to dose the animals lessfrequently in a model of angiogenesis. In mouse Matrigel plug assays,animals were dosed according to a variety of schedules with eitherTM-601 or PEGylated TM-601 (TM-601-PEG). Microvessel densities weremeasured and reduction of such densities was interpreted to signifyanti-angiogenic effects.

Both TM-601 and TM-601-PEG had anti-angiogenic effects with the two mostfrequent dosing schedules tested (twice a week for two weeks, “Q3D×2/2”;and five times a week for two weeks, “Q1D×5/2”) (FIG. 29). WhereasTM-601 did not exhibit any anti-angiogenic effects with the leastfrequent dosing schedule tested (once a week for two weeks, “Q7D×2”)treatment with TM-601-PEG with such a dosing schedule resulted in asignificant reduction of micro-vessel density (FIG. 29).

Without wishing to be bound by any particular theory, the ability todose animals less frequently may be due to availability of TM-601-PEGfor a longer period of time as compared to TM-601. Such increasedavailability could result in longer exposure at sites of new bloodvessel formation, allowing more prolonged effects. These characteristics(e.g., increased availability, prolonged effects, etc.) may beadvantageous in treatment of metastatic tumors.

Example 11 Effect of TM-601 on Lung Metastases

In the present Example, the ability of TM-601 to inhibit metastases inthe lung is investigated using a mouse model in which melanoma cells areinjected into mice. The number of resultant lung metastases in mice withor without treatment with TM-601 is counted.

The B16/F10 mouse melanoma cell line is obtained from ATCC and culturedaccording to recommended specifications. Each mouse is inoculatedintravenously with 0.2 mL of a 0.9% NaCl solution containing asuspension of tumor cells (1×10⁵ cells/mouse).

Drug injections are administered concurrently with tumor cells accordingto Table 1 below. Animals receive doses of test article as described inTable 1 below. Animals receive doses daily five times per week for twoweeks. Sterile PBS are used as the vehicle control. TM-601 arereconstituted in sterile PBS solution. Following treatment initiation,mouse body weight measurements are recorded twice weekly and grossobservations are made at least once daily. All mice are sacrificed fromall groups at Day 14 (or upon determination of moribund state) and thenumber of B16/F10 lung colonies are counted. The counted colonies arevisible as black colored lung nodules on a yellow background visible ininflated lungs with Bouin fixative solution instilled via trachea.

TABLE 1 Evaluation of TM-601 as a Single Agent versus pulmonarymetastasis of B16/F10mouse melanoma Vehicle TM-601 Group Name N (qd × 5× 2) (qd × 5 × 2) 1. Vehicle* 5 X 2. TM-601 2 mg/kg (IV)* 5 X 3. TM-60120 mg/kg (IV)* 5 X *First dose to be administered concurrently w/ tumorcells on Day 0

Other Embodiments

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

What is claimed is:
 1. A method of detecting the presence of one or moremetastases in an individual who has or has had at least one primarytumor, the method comprising steps of: administering to the individualan effective amount of a labeled chlorotoxin agent comprising achlorotoxin polypeptide having at least 90% overall sequence identitywith SEQ ID NO:1, and wherein the labeled chlorotoxin agent selectivelytargets cancer cells over normal cells; wherein the labeled chlorotoxinagent is administered systemically; and, measuring binding of thelabeled chlorotoxin agent in the individual's body in at least onelocation other than the location of the at least one primary tumor,wherein an elevated level of binding, relative to normal tissue,indicates the presence of one or more metastases.
 2. The method of claim1, wherein the labeled chlorotoxin agent is administered intravenously.3. The method of claim 1, wherein the labeled chlorotoxin agent binds toat least one tumor metastasis that is located in the brain.
 4. Themethod of claim 1, wherein the labeled chlorotoxin agent comprises achlorotoxin moiety selected from the group consisting of chlorotoxin,biologically active chlorotoxin subunits, and chlorotoxin derivatives.5. The method of claim 1, wherein the primary tumor is a solid tumor. 6.The method of claim 1, wherein the primary tumor is a refractory tumor.7. The method of claim 1, wherein the primary tumor is a recurrenttumor.
 8. The method of claim 1, wherein the primary tumor is a memberof the group consisting of lung cancer, bone cancer, liver cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,uterine cancer, carcinoma of the sexual or reproductive organs,Hodgkin's Disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, chronic or acute leukemia,lymphocytic lymphomas, cancer of the bladder, cancer of the kidney,renal cell carcinoma, neoplasms of the central nervous system (CNS),neuroectodermal cancer, spinal axis tumors, glioma, meningioma, andpituitary adenoma.
 9. The method of claim 1, wherein the primary tumoris cutaneous or intraocular melanoma.
 10. The method of claim 1, whereinthe primary tumor is a tumor of neuroectodermal origin.
 11. The methodof claim 10, wherein the tumor of neuroectodermal origin is a member ofthe group consisting of glioma, meningioma, ependymoma, medulloblastoma,neuroblastoma, ganglioma, pheochromocytoma, melanoma, peripheralprimitive neuroectodermal tumor, small cell carcinoma of the lung, andEwing's sarcoma.
 12. The method of claim 11, wherein the tumor ofneuroectodermal origin is glioma.
 13. The method of claim 1, wherein thestep of administering comprises systemic administration of at least onedose of labeled chlorotoxin agent.
 14. The method of claim 13, whereinthe step of administering comprising systemic administration of a firstand second doses of labeled chlorotoxin agent, wherein the second doseis higher than the first dose.
 15. The method of claim 13, wherein thestep of administering comprises systemic administration of a first,second and third doses of labeled chlorotoxin agent, wherein the seconddose is higher than the first dose and the third dose is higher than thesecond dose.
 16. The method of claim 1, wherein the labeled chlorotoxinagent is directly detectable.
 17. The method of claim 1, wherein thelabeled chlorotoxin agent is indirectly detectable.
 18. The method ofclaim 1, wherein the labeled chlorotoxin agent comprises at least one ofthe following labeling moieties: radionuclides, fluorophores,chemiluminescent agents, mictoparticles, enzymes, colorimetric labels,magnetic labels, haptens, Molecular Beacons, aptamer beacons, and anycombination thereof.
 19. The method of claim 18 wherein the labelingmoiety is selected from the group consisting of: ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P,³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y, ^(99m)Tc, ¹⁷⁷Lu, ¹³¹I,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ³²P, ¹⁵³Sm, ⁶⁷Ga, ¹¹¹In, ²⁰¹Tl,and any combination thereof.
 20. The method of claim 18, wherein thelabeling moiety is selected from the group consisting of: fluoresceinisothiocyanine or FITC, naphthofluorescein,4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM,carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin,erythrosin, eosin, carboxytetramethyl-rhodamine or TAMRA,carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B,rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine(TMR), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin,aminomethylcoumarin (AMCA), Oregon Green 488, Oregon Green 500, OregonGreen 514, Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™,Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™, Alexa Fluor 350, Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, AlexaFluor 633, Alexa Fluor 660, Alexa Fluor 680, BODIPY FL, BODIPY R6G,BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570,BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, IRD40,IRD 700, IRD 800, and any combination thereof.
 21. The method of claim18, wherein the labeling moiety is selected from the group consistingof: horseradish peroxidase, beta-galactosidase, luciferase, alkalinephosphatase, beta-glucuronidase, beta-D-glucosidase, urease, glucoseoxidase, and any combination thereof.
 22. The method of claim 18,wherein the labeling moiety is selected from the group consisting of:gadolinium III (Gd³⁺), chromium III (Cr³⁺), dysprosium III (Dy³⁺), ironIII (Fe³⁺), manganese II (Mn²⁺), and ytterbium III (Yb³⁺), and anycombination thereof.
 23. The method of claim 1, further comprising astep of administering a chemotherapeutic agent to the individual. 24.The method of claim 23, wherein the chemotherapeutic agent is selectedfrom the group consisting of alkylating agents, purine antagonists,pyrimidine antagonists, plant alkaloids, intercalating antibiotics,aromatase inhibitors, anti-metabolites, mitotic inhibitors, growthfactor inhibitor, cell cycle inhibitors, enzymes, topoisomeraseinhibitors, biological response modifiers, anti-hormones andanti-androgens.
 25. The method of claim 1, wherein the chlorotoxin agentis covalently attached to a polymer.
 26. The method of claim 25, whereinthe polymer is polyethylene glycol (PEG).
 27. The method of claim 1,wherein the chlorotoxin agent is at least 90% identical to an amino acidsequence set forth in SEQ ID NO:
 1. 28. A method of detecting thepresence of one or more metastases in an individual who has or has hadat least one primary tumor, the method comprising steps of:administering to the individual an effective amount of a labeledchlorotoxin agent comprising a chlorotoxin polypeptide as set forth inSEQ ID NO:1, wherein the labeled chlorotoxin agent is administeredsystemically; and measuring binding of the labeled chlorotoxin agent inthe individual's body in at least one location other than the locationof the at least one primary tumor, wherein an elevated level of binding,relative to normal tissue, indicates the presence of one or moremetastases.